Nucleic acid and corresponding protein entitled 161P2F10B useful in treatment and detection of cancer

ABSTRACT

A novel gene 0161P2F10B (also designated 161P2F10B) and its encoded protein, and variants thereof, are described wherein 161P2F10B exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Consequently, 161P2F10B provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The 161P2F10B gene or fragment thereof, or its encoded protein, or variants thereof, or a fragment thereof, can be used to elicit a humoral or cellular immune response; antibodies or T cells reactive with 161P2F10B can be used in active or passive immunization.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/655,822, filed Jan. 19, 2007, now U.S. Pat. No. 7,667,018, issuedFeb. 23, 2010, which is a divisional of U.S. patent application Ser. No.10/291,241, filed Nov. 7, 2002, now U.S. Pat. No. 7,226,594, issued Jun.5, 2007, which is a continuation-in-part of U.S. patent application Ser.No. 10/062,109, filed Jan. 31, 2002, now U.S. Pat. No. 7,067,130, issuedJun. 27, 2006, which is a continuation of U.S. patent application Ser.No. 10/005,480, filed Nov. 7, 2001, now abandoned. The contents of eachapplication listed in this paragraph are fully incorporated by referenceherein for all purposes.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The entire content of the following electronic submission of thesequence listing via the USPTO EFS-WEB server, as authorized and setforth in MPEP §1730 II.B.2(a)(C), is incorporated herein by reference inits entirety for all purposes. The sequence listing is identified on theelectronically filed text file as follows:

File Name Date of Creation Size (bytes) 511582006212Seqlist.txt Feb. 23,2010 286,536 bytes

TECHNICAL FIELD

The invention described herein relates to a gene and its encodedprotein, termed 161P2F10B, expressed in certain cancers, and todiagnostic and therapeutic methods and compositions useful in themanagement of cancers that express 161P2F10B.

BACKGROUND ART

Cancer is the second leading cause of human death next to coronarydisease. Worldwide, millions of people die from cancer every year. Inthe United States alone, as reported by the American Cancer Society,cancer causes the death of well over a half-million people annually,with over 1.2 million new cases diagnosed per year. While deaths fromheart disease have been declining significantly, those resulting fromcancer generally are on the rise. In the early part of the next century,cancer is predicted to become the leading cause of death.

Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the primary causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience physical debilitations following treatment.Furthermore, many cancer patients experience a recurrence.

Worldwide, prostate cancer is the fourth most prevalent cancer in men.In North America and Northern Europe, it is by far the most commoncancer in males and is the second leading cause of cancer death in men.In the United States alone, well over 30,000 men die annually of thisdisease—second only to lung cancer. Despite the magnitude of thesefigures, there is still no effective treatment for metastatic prostatecancer. Surgical prostatectomy, radiation therapy, hormone ablationtherapy, surgical castration and chemotherapy continue to be the maintreatment modalities. Unfortunately, these treatments are ineffectivefor many and are often associated with undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that canaccurately detect early-stage, localized tumors remains a significantlimitation in the diagnosis and management of this disease. Although theserum prostate specific antigen (PSA) assay has been a very useful tool,however its specificity and general utility is widely regarded aslacking in several important respects.

Progress in identifying additional specific markers for prostate cancerhas been improved by the generation of prostate cancer xenografts thatcan recapitulate different stages of the disease in mice. The LAPC (LosAngeles Prostate Cancer) xenografts are prostate cancer xenografts thathave survived passage in severe combined immune deficient (SCID) miceand have exhibited the capacity to mimic the transition from androgendependence to androgen independence (Klein et al., 1997, Nat. Med.3:402). More recently identified prostate cancer markers include PCTA-1(Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252),prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res1996 Sep. 2 (9): 1445-51), STEAP (Hubert, et al., Proc Natl Acad SciUSA. 1999 Dec. 7; 96(25): 14523-8) and prostate stem cell antigen (PSCA)(Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).

While previously identified markers such as PSA, PSM, PCTA and PSCA havefacilitated efforts to diagnose and treat prostate cancer, there is needfor the identification of additional markers and therapeutic targets forprostate and related cancers in order to further improve diagnosis andtherapy.

Renal cell carcinoma (RCC) accounts for approximately 3 percent of adultmalignancies. Once adenomas reach a diameter of 2 to 3 cm, malignantpotential exists. In the adult, the two principal malignant renal tumorsare renal cell adenocarcinoma and transitional cell carcinoma of therenal pelvis or ureter. The incidence of renal cell adenocarcinoma isestimated at more than 29,000 cases in the United States, and more than11,600 patients died of this disease in 1998. Transitional cellcarcinoma is less frequent, with an incidence of approximately 500 casesper year in the United States.

Surgery has been the primary therapy for renal cell adenocarcinoma formany decades. Until recently, metastatic disease has been refractory toany systemic therapy. With recent developments in systemic therapies,particularly immunotherapies, metastatic renal cell carcinoma may beapproached aggressively in appropriate patients with a possibility ofdurable responses. Nevertheless, there is a remaining need for effectivetherapies for these patients.

Of all new cases of cancer in the United States, bladder cancerrepresents approximately 5 percent in men (fifth most common neoplasm)and 3 percent in women (eighth most common neoplasm). The incidence isincreasing slowly, concurrent with an increasing older population. In1998, there was an estimated 54,500 cases, including 39,500 in men and15,000 in women. The age-adjusted incidence in the United States is 32per 100,000 for men and eight per 100,000 in women. The historicmale/female ratio of 3:1 may be decreasing related to smoking patternsin women. There were an estimated 11,000 deaths from bladder cancer in1998 (7,800 in men and 3,900 in women). Bladder cancer incidence andmortality strongly increase with age and will be an increasing problemas the population becomes more elderly.

Most bladder cancers recur in the bladder. Bladder cancer is managedwith a combination of transurethral resection of the bladder (TUR) andintravesical chemotherapy or immunotherapy. The multifocal and recurrentnature of bladder cancer points out the limitations of TUR. Mostmuscle-invasive cancers are not cured by TUR alone. Radical cystectomyand urinary diversion is the most effective means to eliminate thecancer but carry an undeniable impact on urinary and sexual function.There continues to be a significant need for treatment modalities thatare beneficial for bladder cancer patients.

An estimated 130,200 cases of colorectal cancer occurred in 2000 in theUnited States, including 93,800 cases of colon cancer and 36,400 ofrectal cancer. Colorectal cancers are the third most common cancers inmen and women. Incidence rates declined significantly during 1992-1996(−2.1% per year). Research suggests that these declines have been due toincreased screening and polyp removal, preventing progression of polypsto invasive cancers. There were an estimated 56,300 deaths (47,700 fromcolon cancer, 8,600 from rectal cancer) in 2000, accounting for about11% of all U.S. cancer deaths.

At present, surgery is the most common form of therapy for colorectalcancer, and for cancers that have not spread, it is frequently curative.Chemotherapy, or chemotherapy plus radiation, is given before or aftersurgery to most patients whose cancer has deeply perforated the bowelwall or has spread to the lymph nodes. A permanent colostomy (creationof an abdominal opening for elimination of body wastes) is occasionallyneeded for colon cancer and is infrequently required for rectal cancer.There continues to be a need for effective diagnostic and treatmentmodalities for colorectal cancer.

There were an estimated 164,100 new cases of lung and bronchial cancerin 2000, accounting for 14% of all U.S. cancer diagnoses. The incidencerate of lung and bronchial cancer is declining significantly in men,from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s,the rate of increase among women began to slow. In 1996, the incidencerate in women was 42.3 per 100,000.

Lung and bronchial cancer caused an estimated 156,900 deaths in 2000,accounting for 28% of all cancer deaths. During 1992-1996, mortalityfrom lung cancer declined significantly among men (−1.7% per year) whilerates for women were still significantly increasing (0.9% per year).Since 1987, more women have died each year of lung cancer than breastcancer, which, for over 40 years, was the major cause of cancer death inwomen. Decreasing lung cancer incidence and mortality rates most likelyresulted from decreased smoking rates over the previous 30 years;however, decreasing smoking patterns among women lag behind those ofmen. Of concern, although the declines in adult tobacco use have slowed,tobacco use in youth is increasing again.

Treatment options for lung and bronchial cancer are determined by thetype and stage of the cancer and include surgery, radiation therapy, andchemotherapy. For many localized cancers, surgery is usually thetreatment of choice. Because the disease has usually spread by the timeit is discovered, radiation therapy and chemotherapy are often needed incombination with surgery. Chemotherapy alone or combined with radiationis the treatment of choice for small cell lung cancer; on this regimen,a large percentage of patients experience remission, which in some casesis long lasting. There is however, an ongoing need for effectivetreatment and diagnostic approaches for lung and bronchial cancers.

An estimated 182,800 new invasive cases of breast cancer were expectedto occur among women in the United States during 2000. Additionally,about 1,400 new cases of breast cancer were expected to be diagnosed inmen in 2000. After increasing about 4% per year in the 1980s, breastcancer incidence rates in women have leveled off in the 1990s to about110.6 cases per 100,000.

In the U.S. alone, there were an estimated 41,200 deaths (40,800 women,400 men) in 2000 due to breast cancer. Breast cancer ranks second amongcancer deaths in women. According to the most recent data, mortalityrates declined significantly during 1992-1996 with the largest decreasesin younger women, both white and black. These decreases were probablythe result of earlier detection and improved treatment.

Taking into account the medical circumstances and the patient'spreferences, treatment of breast cancer may involve lumpectomy (localremoval of the tumor) and removal of the lymph nodes under the arm;mastectomy (surgical removal of the breast) and removal of the lymphnodes under the arm; radiation therapy; chemotherapy; or hormonetherapy. Often, two or more methods are used in combination. Numerousstudies have shown that, for early stage disease, long-term survivalrates after lumpectomy plus radiotherapy are similar to survival ratesafter modified radical mastectomy. Significant advances inreconstruction techniques provide several options for breastreconstruction after mastectomy. Recently, such reconstruction has beendone at the same time as the mastectomy.

Local excision of ductal carcinoma in situ (DCIS) with adequate amountsof surrounding normal breast tissue may prevent the local recurrence ofthe DCIS. Radiation to the breast and/or tamoxifen may reduce the chanceof DCIS occurring in the remaining breast tissue. This is importantbecause DCIS, if left untreated, may develop into invasive breastcancer. Nevertheless, there are serious side effects or sequelae tothese treatments. There is, therefore, a need for efficacious breastcancer treatments.

There were an estimated 23,100 new cases of ovarian cancer in the UnitedStates in 2000. It accounts for 4% of all cancers among women and rankssecond among gynecologic cancers. During 1992-1996, ovarian cancerincidence rates were significantly declining. Consequent to ovariancancer, there were an estimated 14,000 deaths in 2000. Ovarian cancercauses more deaths than any other cancer of the female reproductivesystem.

Surgery, radiation therapy, and chemotherapy are treatment options forovarian cancer. Surgery usually includes the removal of one or bothovaries, the fallopian tubes (salpingo-oophorectomy), and the uterus(hysterectomy). In some very early tumors, only the involved ovary willbe removed, especially in young women who wish to have children. Inadvanced disease, an attempt is made to remove all intra-abdominaldisease to enhance the effect of chemotherapy. There continues to be animportant need for effective treatment options for ovarian cancer.

There were an estimated 28,300 new cases of pancreatic cancer in theUnited States in 2000. Over the past 20 years, rates of pancreaticcancer have declined in men. Rates among women have remainedapproximately constant but may be beginning to decline. Pancreaticcancer caused an estimated 28,200 deaths in 2000 in the United States.Over the past 20 years, there has been a slight but significant decreasein mortality rates among men (about −0.9% per year) while rates haveincreased slightly among women.

Surgery, radiation therapy, and chemotherapy are treatment options forpancreatic cancer. These treatment options can extend survival and/orrelieve symptoms in many patients but are not likely to produce a curefor most. There is a significant need for additional therapeutic anddiagnostic options for pancreatic cancer.

DISCLOSURE OF THE INVENTION

The present invention relates to a gene, designated 161P2F10B, that hasnow been found to be over-expressed in the cancer(s) listed in Table I.Northern blot expression analysis of 161P2F10B gene expression in normaltissues shows a restricted expression pattern in adult tissues. Thenucleotide (FIG. 2) and amino acid (FIG. 2, and FIG. 3) sequences of161P2F10B are provided. The tissue-related profile of 161P2F10B innormal adult tissues, combined with the over-expression observed in thetissues listed in Table I, shows that 161P2F10B is aberrantlyover-expressed in at least some cancers, and thus serves as a usefuldiagnostic, prophylactic, prognostic, and/or therapeutic target forcancers of the tissue(s) such as those listed in Table I.

The invention provides polynucleotides corresponding or complementary toall or part of the 161P2F10B genes, mRNAs, and/or coding sequences,preferably in isolated form, including polynucleotides encoding161P2F10B-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25contiguous amino acids; at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80,85, 90, 95, 100 or more than 100 contiguous amino acids of a161P2F10B-related protein, as well as the peptides/proteins themselves;DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides oroligonucleotides complementary or having at least a 90% homology to the161P2F10B genes or mRNA sequences or parts thereof, and polynucleotidesor oligonucleotides that hybridize to the 161P2F10B genes, mRNAs, or to161P2F10B-encoding polynucleotides. Also provided are means forisolating cDNAs and the genes encoding 161P2F10B. Recombinant DNAmolecules containing 161P2F10B polynucleotides, cells transformed ortransduced with such molecules, and host-vector systems for theexpression of 161P2F10B gene products are also provided. The inventionfurther provides antibodies that bind to 161P2F10B proteins andpolypeptide fragments thereof, including polyclonal and monoclonalantibodies, murine and other mammalian antibodies, chimeric antibodies,humanized and fully human antibodies, and antibodies labeled with adetectable marker or therapeutic agent. In certain embodiments, there isa proviso that the entire nucleic acid sequence of FIG. 2 is not encodedand/or the entire amino acid sequence of FIG. 2 is not prepared. Incertain embodiments, the entire nucleic acid sequence of FIG. 2 isencoded and/or the entire amino acid sequence of FIG. 2 is prepared,either of which are in respective human unit dose forms.

The invention further provides methods for detecting the presence andstatus of 161P2F10B polynucleotides and proteins in various biologicalsamples, as well as methods for identifying cells that express161P2F10B. A typical embodiment of this invention provides methods formonitoring 161P2F10B gene products in a tissue or hematology samplehaving or suspected of having some form of growth dysregulation such ascancer.

The invention further provides various immunogenic or therapeuticcompositions and strategies for treating cancers that express 161P2F10Bsuch as cancers of tissues listed in Table I, including therapies aimedat inhibiting the transcription, translation, processing or function of161P2F10B as well as cancer vaccines. In one aspect, the inventionprovides compositions, and methods comprising them, for treating acancer that expresses 161P2F10B in a human subject wherein thecomposition comprises a carrier suitable for human use and a human unitdose of one or more than one agent that inhibits the production orfunction of 161P2F10B. Preferably, the carrier is a uniquely humancarrier. In another aspect of the invention, the agent is a moiety thatis immunoreactive with 161P2F10B protein. Non-limiting examples of suchmoieties include, but are not limited to, antibodies (such as singlechain, monoclonal, polyclonal, humanized, chimeric, or humanantibodies), functional equivalents thereof (whether naturally occurringor synthetic), and combinations thereof. The antibodies can beconjugated to a diagnostic or therapeutic moiety. In another aspect, theagent is a small molecule as defined herein.

In another aspect, the agent comprises one or more than one peptidewhich comprises a cytotoxic T lymphocyte (CTL) epitope that binds an HLAclass I molecule in a human to elicit a CTL response to 161P2F10B and/orone or more than one peptide which comprises a helper T lymphocyte (HTL)epitope which binds an HLA class II molecule in a human to elicit an HTLresponse. The peptides of the invention may be on the same or on one ormore separate polypeptide molecules. In a further aspect of theinvention, the agent comprises one or more than one nucleic acidmolecule that expresses one or more than one of the CTL or HTL responsestimulating peptides as described above. In yet another aspect of theinvention, the one or more than one nucleic acid molecule may express amoiety that is immunologically reactive with 161P2F10B as describedabove. The one or more than one nucleic acid molecule may also be, orencodes, a molecule that inhibits production of 161P2F10B. Non-limitingexamples of such molecules include, but are not limited to, thosecomplementary to a nucleotide sequence essential for production of161P2F10B (e.g. antisense sequences or molecules that form a triplehelix with a nucleotide double helix essential for 161P2F10B production)or a ribozyme effective to lyse 161P2F10B mRNA.

Note that to determine the starting position of any peptide set forth inTables VIII-XXI and XXII to XLIX (collectively HLA Peptide Tables)respective to its parental protein, e.g., variant 1, variant 2, etc.,reference is made to three factors: the particular variant, the lengthof the peptide in an HLA Peptide Table, and the Search Peptides in TableVII. Generally, a unique Search Peptide is used to obtain HLA peptidesof a particular for a particular variant. The position of each SearchPeptide relative to its respective parent molecule is listed in TableVII. Accordingly, if a Search Peptide begins at position “X”, one mustadd the value “X−1” to each position in Tables VIII-XXI and XXII to XLIXto obtain the actual position of the HLA peptides in their parentalmolecule. For example, if a particular Search Peptide begins at position150 of its parental molecule, one must add 150−1, i.e., 149 to each HLApeptide amino acid position to calculate the position of that amino acidin the parent molecule.

One embodiment of the invention comprises an HLA peptide, that occurs atleast twice in Tables VIII-XXI and XXII to XLIX collectively, or anoligonucleotide that encodes the HLA peptide. Another embodiment of theinvention comprises an HLA peptide that occurs at least once in TablesVIII-XXI and at least once in tables XXII to XLIX, or an oligonucleotidethat encodes the HLA peptide.

Another embodiment of the invention is antibody epitopes, which comprisea peptide regions, or an oligonucleotide encoding the peptide region,that has one two, three, four, or five of the following characteristics:

i) a peptide region of at least 5 amino acids of a particular peptide ofFIG. 3, in any whole number increment up to the full length of thatprotein in FIG. 3, that includes an amino acid position having a valueequal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a valueequal to 1.0, in the Hydrophilicity profile of FIG. 5;

ii) a peptide region of at least 5 amino acids of a particular peptideof FIG. 3, in any whole number increment up to the full length of thatprotein in FIG. 3, that includes an amino acid position having a valueequal to or less than 0.5, 0.4, 0.3, 0.2, 0.1, or having a value equalto 0.0, in the Hydropathicity profile of FIG. 6;

iii) a peptide region of at least 5 amino acids of a particular peptideof FIG. 3, in any whole number increment up to the full length of thatprotein in FIG. 3, that includes an amino acid position having a valueequal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a valueequal to 1.0, in the Percent Accessible Residues profile of FIG. 7;

iv) a peptide region of at least 5 amino acids of a particular peptideof FIG. 3, in any whole number increment up to the full length of thatprotein in FIG. 3, that includes an amino acid position having a valueequal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a valueequal to 1.0, in the Average Flexibility profile of FIG. 8; or

v) a peptide region of at least 5 amino acids of a particular peptide ofFIG. 3, in any whole number increment up to the full length of thatprotein in FIG. 3, that includes an amino acid position having a valueequal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a valueequal to 1.0, in the Beta-turn profile of FIG. 9.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The 161P2F10B SSH sequence of 182 nucleotides.

FIG. 2. A) The cDNA and amino acid sequence of 161P2F10B variant 1 (alsocalled “161P2F10B v.1” or “161P2F10B variant 1”) is shown in FIG. 2A.The start methionine is underlined. The open reading frame extends fromnucleic acid 44-2671 including the stop codon.

B) The cDNA and amino acid sequence of 161P2F10B variant 2 (also called“161P2F10B v.2”) is shown in FIG. 2B. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 44-2671including the stop codon.

C) The cDNA and amino acid sequence of 161P2F10B variant 3 (also called“161P2F10B v.3”) is shown in FIG. 2C. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 44-2671including the stop codon. The cDNA and amino acid sequence of 161P2F10Bvariant 4 (also called “161P2F10B v.4”) is shown in D) FIG. 2D. Thecodon for the start methionine is underlined. The open reading frameextends from nucleic acid 44-2671 including the stop codon.

E) The cDNA and amino acid sequence of 161P2F10B variant 5 (also called“161P2F10B v.5”) is shown in FIG. 2E. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 44-2671including the stop codon.

F) The cDNA and amino acid sequence of 161P2F10B variant 6 (also called“161P2F10B v.6”) is shown in FIG. 2F. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 84-2711including the stop codon.

G) The cDNA and amino acid sequence of 161P2F10B variant 7 (also called“161P2F10B v.7”) is shown in FIG. 2G. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 276-2801including the stop codon.

FIG. 3.

A) Amino acid sequence of 161P2F10B v.1 is shown in FIG. 3A; it has 875amino acids.

B) The amino acid sequence of 161P2F10B v.2 is shown in FIG. 3B; it has875 amino acids.

C) The amino acid sequence of 161P2F10B v.3 is shown in FIG. 3C; it has875 amino acids.

D) The amino acid sequence of 161P2F10B v.4 is shown in FIG. 3D; it has875 amino acids.

E) The amino acid sequence of 161P2F10B v.7 is shown in FIG. 3E; it has841 amino acids. As used herein, a reference to 161P2F10B includes allvariants thereof, including those shown in FIGS. 2, 3, 10, and 11,unless the context clearly indicates otherwise.

FIG. 4. FIG. 4A: Alignment of 161P2F10 with variant 1 carrying a K to Rmutation. FIG. 4B: Alignment of 161P2F10B and SNP variant carrying a Tto P mutation.

FIG. 5. Hydrophilicity amino acid profile of 161P2F10B determined bycomputer algorithm sequence analysis using the method of Hopp and Woods(Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A.78:3824-3828) accessed on the Protscale website located on the WorldWide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasymolecular biology server.

FIG. 6. Hydropathicity amino acid profile of 161P2F10B determined bycomputer algorithm sequence analysis using the method of Kyte andDoolittle (Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132)accessed on the ProtScale website located on the World Wide Web throughthe ExPasy molecular biology server.

FIG. 7. Percent accessible residues amino acid profile of 161P2F10Bdetermined by computer algorithm sequence analysis using the method ofJanin (Janin J., 1979 Nature 277:491-492) accessed on the ProtScalewebsite located on the World Wide Web through the ExPasy molecularbiology server.

FIG. 8. Average flexibility amino acid profile of 161P2F10B determinedby computer algorithm sequence analysis using the method of Bhaskaranand Ponnuswamy (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept.Protein Res. 32:242-255) accessed on the ProtScale website located onthe World Wide Web through the ExPasy molecular biology server.

FIG. 9. Beta-turn amino acid profile of 161P2F10B determined by computeralgorithm sequence analysis using the method of Deleage and Roux(Deleage, G., Roux B. 1987 Protein Engineering 1:289-294) accessed onthe ProtScale website located on the World Wide Web through the ExPasymolecular biology server.

FIG. 10. Variants 161P2F10B v.2 through v.5 are variants with singlenucleotide differences. Though these SNP variants are shown separately,they could also occur in any combinations and in any transcript variantsthat contains the base pairs. Variants 161P2F10B v.6 and v.7 aretranscript variants. Variant 161P2F10B v.6 has extra 40 bases at the 5′end and a different 3′ end portion, while variant 161P2F10B v.7 has aninsertion of 130 bases in between positions 121 and 122 of 161P2F10Bv.1. Numbers in “( )” correspond to those of 161P2F10B v.1. Black boxshows the same sequence as 161P2F10B v.1. SNPs are indicated above thebox.

FIG. 11. Protein variants correspond to nucleotide variants. Nucleotidevariants 161P2F10B v.5 and v.6 in FIG. 10 code for the same protein as161P2F10B v.1. Nucleotide variants 161P2F10B v.6 and v.7 are splicevariants of v.1, as shown in FIG. 12. Single amino acid differences wereindicated above the boxes. Black boxes represent the same sequence as161P2F10B v.1. Numbers underneath the box correspond to 161P2F10B v.1.

FIG. 12. The secondary structure of 161P2F10B (SEQ ID NO: 103), namelythe predicted presence and location of alpha helices, extended strands,and random coils, is predicted from the primary amino acid sequenceusing the HNN—Hierarchical Neural Network method, accessed from theExPasy molecular biology server on the World Wide Web. The analysisindicates that 161P2F10B is composed 31.31% alpha helix, 11.31% extendedstrand, and 57.37% random coil.

FIG. 13. Shown graphically in FIG. 13A-B are the results of analysisusing the TMpred (FIG. 13A) and TMHMM (FIG. 13B) prediction programsdepicting the location of the transmembrane domain.

FIG. 14. First strand cDNA was generated from normal stomach, normalbrain, normal heart, normal liver, normal skeletal muscle, normaltestis, normal prostate, normal bladder, normal kidney, normal colon,normal lung, normal pancreas, and a pool of cancer specimens fromprostate cancer patients, bladder cancer patients, kidney cancerpatients, colon cancer patients, lung cancer patients, pancreas cancerpatients, a pool of prostate cancer xenografts (LAPC-4AD, LAPC-4AI,LAPC-9AD and LAPC-9AI), and a pool of 2 patient prostate metastasis tolymph node. Normalization was performed by PCR using primers to actin.Semi-quantitative PCR, using primers to 161P2F10B, was performed at 26and 30 cycles of amplification. Samples were run on an agarose gel, andPCR products were quantitated using the AlphaImager software. Resultsshow strong expression in prostate cancer, bladder cancer, kidneycancer, colon cancer, lung cancer, pancreas cancer, bone cancer,lymphoma cancer, uterus cancer, compared to all normal tissues tested.Strong expression was also detected in the xenograft pool as well as theprostate cancer metastasis to lymph node specimens.

FIG. 15. First strand cDNA was prepared from a panel of kidney cancerclear cell carcinoma (A), kidney cancer papillary carcinoma (B), and inuterus patient cancer specimens (C). Normalization was performed by PCRusing primers to actin. Semi-quantitative PCR, using primers to161P2F10B, was performed at 26 and 30 cycles of amplification. Sampleswere run on an agarose gel, and PCR products were quantitated using theAlphaImager software. Expression was recorded as absent, low, medium orstrong. Results show expression of 161P2F10B in 94.7% of clear cellrenal carcinoma, 62.5% of papillary renal cell carcinoma, and in 61.5%of uterus cancer.

FIG. 16. Shows Phosphodiesterase Activity of 3T3-161P2F10B Stable Cells.Cell surface phosphodiesterase activity is assayed on 3T3 and3T3-161P2F10B using the substrate p-nitrophenyl thymidine-5′-L-monosphosphate.

FIG. 17. Shows Protection from Apoptosis by 161P2F10B.

FIG. 18. Shows that 161P2F10B Protects from Apoptotic Signals.

FIG. 19. Shows that 161P2F10B Protects from Staurosporine and UV-InducedApoptosis.

FIG. 20. Shows that 161P2F10B Expression Protects Cells from Drug andUV-Induced Apoptosis. NIH 3T3 cells were treated with the staurosporineor UV, stained with Annexin V-FITC and propidium iodide, and analyzed byFACS.

FIG. 21. Shows that 161P2F10B Protects from Apoptosis byChemotherapeutic Agents.

FIG. 22 Shows the effect of 161P2F10B on In Vitro Invasion. Invasion wasdetermined by measuring the fluorescence of cells in the lower chamberrelative to the fluorescence of the entire cell population.

FIG. 23. Shows that 161P2F10B MAb Attenuates the Growth of Human KidneyCancer Xenograft in SCID Mice.

FIG. 24. Detection of 161P2F10B protein by immunohistochemistry inkidney cancer patient specimens. Two renal clear cell carcinoma tissuespecimens and one renal papillary cell carcinoma were obtained fromthree different kidney cancer patients. Frozen tissues were cut into 4micron sections and fixed in acetone for 10 minutes. The sections werethen incubated with mouse monoclonal anti-ENPP3 antibody(Coulter-Immunotech, Marseilles, France) for 3 hours. The slides werewashed three times in buffer, and further incubated with DAKO EnVision+™peroxidase-conjugated goat anti-mouse secondary antibody (DAKOCorporation, Carpenteria, Calif.) for 1 hour. The sections were thenwashed in buffer, developed using the DAB kit (SIGMA Chemicals),counterstained using hematoxylin, and analyzed by bright fieldmicroscopy. The results showed strong expression of 161P2F10B in allthree renal carcinoma patient tissues (FIG. 24 panels A-C). Theexpression was detected mostly around the cell membrane in the renalclear cell carcinoma specimens, indicating that 161P2F10B is membraneassociated in this kidney cancer, and throughout the cells in thepapillary cell carcinoma with an apparent predisposition towards thecell periphery.

FIG. 25. Detection of 161P2F10B protein by immunohistochemistry in aprostate cancer patient specimen. Tissue specimens of prostateadenocarcinoma were obtained from eight different prostate cancerpatients. Frozen tissues were cut into 4 micron sections and fixed inacetone for 10 minutes. The sections were then incubated with mousemonoclonal anti-ENPP3 antibody (Coulter-Immunotech, Marseilles, France)for 3 hours. The slides were washed three times in buffer, and furtherincubated with DAKO EnVision+™ peroxidase-conjugated goat anti-mousesecondary antibody (DAKO Corporation, Carpenteria, Calif.) for 1 hour.The sections were then washed in buffer, developed using the DAB kit(SIGMA Chemicals), counterstained using hematoxylin, and analyzed bybright field microscopy. The results showed expression of 161P2F10B insix of the eight prostate cancer patient tissues, one of which isillustrated in this FIG. 25. 161P2F10B was expressed on the tumor cellswith an apparent proclivity towards the luminal cell surface.

FIG. 26. Detection of 161P2F10B protein by immunohistochemistry in acolon cancer patient specimen. Tissue specimens of colon adenocarcinomawere obtained from nine different colon cancer patients. Frozen tissueswere cut into 4 micron sections and fixed in acetone for 10 minutes. Thesections were then incubated with mouse monoclonal anti-ENPP3 antibody(Coulter-Immunotech, Marseilles, France) for 3 hours. The slides werewashed three times in buffer, and further incubated with DAKO EnVision+™peroxidase-conjugated goat anti-mouse secondary antibody (DAKOCorporation, Carpenteria, Calif.) for 1 hour. The sections were thenwashed in buffer, developed using the DAB kit (SIGMA Chemicals),counterstained using hematoxylin, and analyzed by bright fieldmicroscopy. The results showed strong expression of 161P2F10B in two ofthe nine colon cancer patient tissues, one of which is illustrated inthis FIG. 26. 161P2F10B was most strongly expressed on the tumor cellswith a luminal cell surface but was also expressed throughout all thetumor tissue.

FIG. 27. Detection by immunohistochemistry of 161P2F10B proteinexpression in kidney clear cell cancer patient specimens by specificbinding of mouse monoclonal antibodies. Renal clear cell carcinomatissue and its matched normal adjacent were obtained from a kidneycancer patient. Frozen tissues were cut into 4 micron sections and fixedin acetone for 10 minutes. The sections were then incubated either mousemonoclonal anti-ENPP3 antibody (Coulter-Immunotech, Marseilles, France)for 3 hours (FIG. 27 panels A, D), or mouse monoclonal antibody X41(3)50(FIG. 27 panels B, E), or mouse monoclonal antibody X41(3)37 (FIG. 27panels C, F). The slides were washed three times in buffer and furtherincubated with DAKO EnVision+™ peroxidase-conjugated goat anti-mousesecondary antibody (DAKO Corporation, Carpenteria, Calif.) for 1 hour.The sections were then washed in buffer, developed using the DAB kit(SIGMA Chemicals), counterstained using hematoxylin, and analyzed bybright field microscopy (FIG. 27 panels A-F). The results showed strongexpression of 161P2F10B in the renal clear cell carcinoma patient tissue(FIG. 27 panels A-C), but weakly in normal kidney (FIG. 27 panels D-F).The expression was predominantly around the cell periphery indicatingthat 161P2F10B is membrane associated in kidney cancer tissues. The weakexpression detected in normal kidney was localized to the kidneyproximal tubules.

FIG. 28. Expression of 161P2F10b in recombinant cell lines.

A.) Rat1, NIH3T3, NSO, and 300.19 cells stably expressing either16P2F10b or a control vector (neo) were stained with PE-conjugatedanti-CD203c MAb and examined by flow cytometry. (Light dotted line:control neo cells. Dark line: 161P2F10 cells)

B.) Rat1, NIH3T3, NSO, 300.19, and UT7 cells were stained with eitherPE-conjugated anti-CD203c MAb or control IgG1-PE Ab and examined by flowcytometry. (Light dotted line: control MAb. Dark line: 97A6 (CD203c)MAb.) Shown is the mean fluorescence of the staining of the control and161P2F10b cells and the ratio of the values. This was used to rank thecells for relative expression levels of 161P2F10b.

C.) The relative cell surface phosphodiesterase enzymatic activity ofthe recombinant cells was measured by the addition of p-nitrophenylthymidine-5′-L-monophosphate (p-nTMP) phosphodiesterase substrate. Thereis a correlation between expression levels determined by flow cytometryand surface enzyme activity.

FIG. 29. Surface expression and phosphodiesterase activity of 161P2F10b.

A. 161P2F10b transfected 293T cells were stained with the commerciallyavailable (Coulter Immunotech) PE-conjugated anti-CD-203c MAb, acommercially available anti-ENPP3 (161P2F10b) MAb and examined byfluorescent microscopy.

B. 161P2F10b and vector transfected 293T cells were incubated in assaybuffer containing the phosphodiesterase-1 colorimetic substratep-nitrophenyl thymidine-5′-L-monophosphate (p-nTMP) and opticaldensities (O.D.) were obtained at 405 nm

FIG. 30. Relative expression and enzymatic activity of 161P2F10b mutantsin recombinant Caki kidney cancer cells. Caki kidney cancer cells wereinfected with retrovirus containing either wildtype 161P2F120b cDNA, orpoint mutant cDNAs encoding either a threonine to serine mutation (T/S)at amino acid 205, a threonine to alanine mutation (T/A) at amino acid205, or a aspartic acid to glutamic acid mutation (D/E) at amino acid80. Stably expressing cell lines were analyzed for 161P2F10b expressionby flow cytometry with 97A6 (CD203c) MAb (A) and for enzymatic activitywith p-nTMP substrate (B). Mutation of threonine 205 to aspartic acid oralanine abolishes the ability to cleave the substrate, demonstratingthat threonine 205 is critical to the enzymatic activity of 161P2F10b.

FIG. 31. Purification of a recombinant protein encoding theextracellular domain (ECD) of 161P2F10b. 293T cells were transfectedwith a Tag5 secretion expression vector encoding the ECD of 161P2F10b(amino acids 46-875). The recombinant protein was purified from theconditioned media using either metal chelate affinity chromatography(not shown) or with an immunoaffinity column comprised of anti-161P2F10bMAb X41.6 (shown). 2 ul of 2 separate purified lots were analyzed bySDS-PAGE and Coomasie staining. BSA protein was also analyzed as aquantitative standard.

FIG. 32. 161P2F10b enzymatic assays utilizing P-nitrophenyl-thymidinemonophosphate (p-nTMP).

A. Schematic of the colorimetric enzyme assay showing enzymatic cleavageof the p-nTMP substrate generating a soluble yellow product.

B. Kinetics and dose response of the enzymatic action of purifiedTag5-ECD 161P2F10b protein on p-nTMP (2.5 mM). Optical densities (OD) ofreactions were determined at 405 nm

C. Cell surface enzymatic assay of 161P2F10b-expressing Rat1 cells. Theindicated number of Rat1-161P2F10b cells were incubated with p-nTMPsubstrate and the OD's of the wells were determined.

D. ATP and NAD (not shown) serve as competitive inhibitors 161P2F10bcleavage of p-nTMP. Purified Tag5-ECD protein (20 ng) was incubated withp-nTMP substrate in the absence or presence of the indicated amounts ofATP. The OD's of reactions were obtained at 405 nm.

FIG. 33. Analysis of the internalization of anti-161P2F10b MAb X41.6.

Panel A. Schematic of the protocol. Rat1-161P2F10b cells are incubatedwith anti-161P2F10b MAb at 4 C, washed, and then either kept at 4 C andstained with anti-mouse IgG secondary-PE conjugated Ab at 4 C (B, totalsurface staining) or moved to 37 C for various times and then stainedwith secondary Ab at 4 C (C, residual surface staining). Panels B and Cdemonstrate that MAb X41.6 engagement of surface 161P2F10b causesinternalization at 37 C of the complex indicated by the progressivedecrease in mean fluorescence intensity (MFI).

FIG. 34. Internalization of selected anti-161P2F10b murine MAbs.Internalization of selected anti-161P2F10b MAbs are by flow cytometryare shown. Internalization is indicated by a decrease in the meanfluorescence intensity (MFI) of cells moved to 37 C versus cells stainedat 4 C.

FIG. 35. Antibody engagement of 161P2F10b results in itsinternalization. Internalization of the commercially available MAb 97A6,anti-CD203c, is shown by fluorescence microscopy following staining ofRat1-161P2F10b cells. The cells were incubated with CD203c-PE conjugatedMAb at 4 C, washed, and then moved to 37 C for the indicated times andthen examined by fluorescence microscopy. At 4 C, the staining of thecells is cell surface (bright halo of fluorescence around individualcells). Upon moving to 37 C, there is a gradual loss of the surfacefluorescence, concomitant with capping of the MAb to punctate regions onthe surface, followed by the appearance of punctate and diffuseintracellular fluorescence and a total loss of surface fluorescence.

FIG. 36. Effects of X41.50 MAb-saporin toxin conjugate on Caki-161P2F10bcells. Shown is the morphology of Caki-161P2F10b cells that were treatedwith and without the indicated concentrations of the internalizinganti-161P2F10b MAb and an anti-mouse IgG-saporin toxin secondary Ab (2ug/ml). Saporin is unable to enter cells efficiently on its own and mustbe internalized for its toxic mechanism (protein synthesis inhibition)to work. Cells were first incubated at 4 C with X41.50 MAb to allowsurface binding, than either media or the saporin-conjugated secondaryAb was added and the cells were incubated for 72 hours at 37 C. Cellsincubated with either media alone, X41.50 alone, or thesecondary-saporin Ab alone had no effect on Caki-161P2F10b growth andmorphology, exemplified by a viable confluent monolayer. However, cellsincubated with X41.50 MAb (2 and 0.5 ug/ml) and the secondarysaporin-conjugate exhibited signs of growth inhibition (did not reachconfluency) and apoptosis (small round floating apoptotic cells abovethe attached cell layer). This demonstrates the utility ofanti-161P2F10b MAbs drug/toxin conjugates as a therapeutic approach for161P2F10b-expressing cancers and diseased tissues.

FIG. 37. Internalization-mediated downregulation of 161P2F10 protein byMAb X41.50. Rat1-161P2F10b cells were incubated with and without 10ug/ml of MAb X41.50 for 72 hours. Cells were washed, fixed,permeabilized, and stained with PE-conjugated CD203c MAb to monitortotal 161P2F10b protein expression. The data shows a marked decrease instaining following treatment of the cells with X41.50, demonstratingdownregulation of 161P2F10b protein.

FIG. 38. Anti-161P2F10b MAbs downregulate surface 161P2F10b enzymaticactivity. Rat1-161P2F10b cells were treated with and without variousconcentrations of the indicated MAbs for 48 hours and then assayed forsurface enzymatic activity using p-n-TMB substrate. The datademonstrates that engagement and internalization of surface 161P2F10b byMAbs results in the concamitant loss of surface 161P2F10b enzymaticactivity.

FIG. 39. Characteristics of mouse 161P2F10b MAbs. Shown is a summary ofvarious characteristics of MAbs that recognize 161P2F10b.

The relative affinity of the MAbs was determined by saturation bindingELISA using the recombinant Tag5-ECD protein. The Kd of the bindingreaction was determined using a one-site binding non-linear regressionanalysis of the data using GraphPad Prism software version 3.02(Graphpad Software, San Diego, Calif.).

Relative surface staining was determined using 10 ug/ml of each MAb onRAT1-161P2F10b cells.

Relative ability to internalize was also carried out on Rat1-161P2F10bcells comparing staining with 10 ug/ml of MAb at 4 C versus residualstaining following incubation at 37 C for 30 minutes.

The ability of the MAbs to downregulate surface enzyme activity wasdetermined by incubation of Rat1-161P2F10b cells with 10 ug/ml of eachMAb for 72 hours then assaying surface enzyme activity with p-nTMPsubstrate.

Relative specific immunohistochemical staining (IHC) was determinedusing 10 ug/ml of each MAb on 161P2F10b-expressing frozen section kidneyclear cell carcinoma samples.

The epitope family was determined by competition binding ELISA using theTag5-ECD protein as target. Tag5-ECD ELISA coated wells were firstincubated with or without 10 ug/ml of competitor MAb, washed, and thenincubated with 1 ug/ml of HRP-labeled test MAb. MAb that compete forbinding (reduction of the signal of the test MAb with prior incubationwith competitor) must share the same or an overlapping epitope and arethus assigned to an epitope family. Of the MAbs listed, at least 2epitope families are defined.

FIG. 40. Surface staining of selected anti-161P2F10b MAbs. Specificbinding of cell surface 161P2F10b was determined by incubation ofRat1-161P2F10 (dark line) and Rat1-neo control cells (light dotted line)with 10 ug/ml of each MAb for 1.5 hours at 4 C. Cells were washed,incubated with goat-anti-mouse-PE conjugated secondary Ab, washed again,and analyzed by flow cytometry. Shown are examples of MAb derived fromDNA-based immunization of mice with an FC-fusion of the ECD (X41.6,X41.15, X41.17, X41.29, X41.37, X41.50), also DNA-based immunizationwith Tag5-ECD, and with Rat1-161P2F10b cells (the last data wasgenerated using the respective hybridoma supernatant at a 1:50 dilution)was performed.

FIG. 41. Anti-161P2F10b MAbs X41.6 and 97A6 (CD203c) do not cross-reactwith ENPP1. Conditioned media from 293T cells transfected with eitherTag5-161P2F10b or ENPP1 His-tagged vectors was subjected toimmunoprecipitation analysis using 5 ug of MAb X41.6, MAb 97A6 (CD203c),or anti-His pAb. Following washing of the immune complexes,phosphodiesterase activity was determined by the addition of p-nTMPsubstrate. Enzymatic activity is seen in anti-His immune complexes fromboth Tag5 161P2F10b and Tag5 ENPP1 media due to the presence of the Hisepitope in both proteins. However, enzymatic activity is seen only inthe immune complexes of X41.6 and 97A6 from Tag5 161P2F10 conditionedmedia and not with Tag5 ENPP1 media. These data demonstrate that MAbsX41.6 and 97A6 (CD203c) do not crossreact with the homologousectonucleotide pyrophosphatase/phosphodiesterase family member ENPP1.

FIG. 42. Detection of 161P2F10b in the conditioned media of161P2F10b-expressing cells. Supernatants of the indicated161P2F20b-expressing and non-expressing cell lines were analyzed forshedding/secretion of 161P2F10b protein by a capture ELISA. The captureELISA was made using a 161P2F10b-specific MAb as the bottom capture MAb(1 ug/well), and X41.29 as the top detection MAb (2 ug/ml), and ananti-mouse IgG2a-HRP secondary and tetramethylbenzamidine as substratefor development. Recombinant 161P2F10b Tag5 ECD protein was used as astandard. 161P2F10b protein was detected in the media from 769 and Cakikidney cancer cells engineered to express 161P2F10b but not in theparental lines, indicating that 161P2F10b protein is shed or secreted.Shed/secreted 161P2F10b may exert its activity on cells in anautocrine/paracrine manner. In addition, shed/secreted 161P2F10b isuseful as a diagnostic marker for 161P2F10b-expressing cancer and/orother 161P2F10b-expressing diseased tissues.

FIG. 43. Detection of secreted 161P2F10B in the serum of mice bearingUGK3 human kidney cancer xenografts. SCID mice inoculated subcutaneouslywith UGK3 kidney cancer cells were monitored for tumor growth (1dimensional tumor measurements) and 161P2F10b serum levels (by captureELISA) over the indicated times. The data demonstrates that 161P2F10bserum levels increase as the tumor size increases. This demonstratesthat 161P2F10b is shed/secreted from 161P2F10b-expressing tissues invivo and further demonstrates the utility of an ELISA to monitor161P2F10b as a diagnostic marker.

FIG. 44: Detection of 161P2F10B protein by immunohistochemistry inkidney cancer patient specimens. Renal clear cell carcinoma tissue andits matched normal adjacent tissue as well as its metastatic cancer tolymph node were obtained from a kidney cancer patient. Frozen tissueswere cut into 4 micron sections and fixed in acetone for 10 minutes. Thesections were then incubated with PE-labeled mouse monoclonal anti-ENPP3antibody (Coulter-Immunotech, Marseilles, France) for 3 hours (FIG. 44panels A-F), or isotype control antibody (FIG. 44 panels G-I). Theslides were washed three times in buffer, and either analyzed byfluorescence microscopy (FIG. 44 panels A, B and C), or furtherincubated with DAKO EnVision+™ peroxidase-conjugated goat anti-mousesecondary antibody (DAKO Corporation, Carpenteria, Calif.) for 1 hour(FIG. 44 panels D, E, and F). The sections were then washed in buffer,developed using the DAB kit (SIGMA Chemicals), counterstained usinghematoxylin, and analyzed by bright field microscopy (FIG. 44 panels D,E and F). The results showed strong expression of 161P2F10B in the renalcarcinoma patient tissue (FIG. 44 panels A and D) and the kidney cancermetastasis to lymph node tissue (FIG. 44 panels C and F), but weakly innormal kidney (FIGS. 44B and E). The expression was detected mostlyaround the cell membrane indicating that 161P2F10B is membraneassociated in kidney cancer tissues. The weak expression detected innormal kidney was localized to the kidney tubules. The sections stainedwith the isotype control antibody were negative showing the specificityof the anti-ENPP3 antibody (FIG. 44 panels G-I).

FIG. 45: Expression of 161P2F10B in Human Patient Cancers by WesternBlot. Cell lysates from kidney cancer tissues (KiCa), kidney cancermetastasis to lymph node (KiCa Met), as well as normal kidney (NK) weresubjected to Western analysis using an anti-161P2F10B mouse monoclonalantibody. Briefly, tissues (˜25 μg total protein) were solubilized inSDS-PAGE sample buffer and separated on a 10-20% SDS-PAGE gel andtransferred to nitrocellulose. Blots were blocked in Tris-bufferedsaline (TBS)+3% non-fat milk and then probed with purifiedanti-161P2F10B antibody in TBS+0.15% Tween-20+1% milk Blots were thenwashed and incubated with a 1:4,000 dilution of anti-mouse IgG-HRPconjugated secondary antibody. Following washing, anti-161P2F10Bimmunoreactive bands were developed and visualized by enhancedchemiluminescence and exposure to autoradiographic film. The specificanti-161P2F10B immunoreactive bands represent a monomeric form of the161P2F10B protein, which runs at approximately 130 kDa. These resultsdemonstrate that 161P2F10B is useful as a diagnostic and therapeutictarget for kidney cancers, metastatic cancers and other such as those aslisted in Table I and other human cancers that express 161P2F10B.

FIG. 46: Expression of 161P2F10B in Human Xenograft Tissues by WesternBlot. Cell lysates from kidney cancer xenograft (KiCa Xeno), kidneycancer metastasis to lymph node xenograft (Met Xeno), as well as normalkidney (NK) were subjected to Western analysis using an anti-161P2F10Bmouse monoclonal antibody. Briefly, tissues (˜25 μg total protein) weresolubilized in SDS-PAGE sample buffer and separated on a 10-20% SDS-PAGEgel and transferred to nitrocellulose. Blots were blocked inTris-buffered saline (TBS)+3% non-fat milk and then probed with purifiedanti-161P2F10B antibody in TBS+0.15% Tween-20+1% milk Blots were thenwashed and incubated with a 1:4,000 dilution of anti-mouse IgG-HRPconjugated secondary antibody. Following washing, anti-161P2F10Bimmunoreactive bands were developed and visualized by enhancedchemiluminescence and exposure to autoradiographic film. The specificanti-161P2F10B immunoreactive bands represent a monomeric form of the161P2F10B protein, which runs at approximately 130 kDa, and a multimerof approximately 260 kDa. These results demonstrate that the humancancer xenograft mouse models can be used to study the diagnostic andtherapeutic effects of 161P2F10B.

DETAILED DESCRIPTION OF THE INVENTION

Outline of Sections

-   -   I.) Definitions    -   II.) 161P2F10B Polynucleotides        -   II.A.) Uses of 161P2F10B Polynucleotides            -   II.A.1.) Monitoring of Genetic Abnormalities            -   II.A.2.) Antisense Embodiments            -   II.A.3.) Primers and Primer Pairs            -   II.A.4.) Isolation of 161P2F10B-Encoding Nucleic Acid                Molecules            -   II.A.5.) Recombinant Nucleic Acid Molecules and                Host-Vector Systems    -   III.) 161P2F10B-related Proteins        -   III.A.) Motif-bearing Protein Embodiments        -   III.B.) Expression of 161P2F10B-related Proteins        -   III.C.) Modifications of 161P2F10B-related Proteins        -   III.D.) Uses of 161P2F10B-related Proteins    -   IV.) 161P2F10B Antibodies    -   V.) 161P2F10B Cellular Immune Responses    -   VI.) 161P2F10B Transgenic Animals    -   VII.) Methods for the Detection of 161P2F10B    -   VIII.) Methods for Monitoring the Status of 161P2F10B-related        Genes and Their Products    -   IX.) Identification of Molecules That Interact With 161P2F10B    -   X.) Therapeutic Methods and Compositions        -   X.A.) Anti-Cancer Vaccines        -   X.B.) 161P2F10B as a Target for Antibody-Based Therapy        -   X.C.) 161P2F10B as a Target for Cellular Immune Responses            -   X.C.1. Minigene Vaccines            -   X.C.2. Combinations of CTL Peptides with Helper Peptides            -   X.C.3. Combinations of CTL Peptides with T Cell Priming                Agents            -   X.C.4. Vaccine Compositions Comprising DC Pulsed with                CTL and/or HTL Peptides            -   X.D.) Adoptive Immunotherapy            -   X.E.) Administration of Vaccines for Therapeutic or                Prophylactic Purposes    -   XI.) Diagnostic and Prognostic Embodiments of 161P2F10B.    -   XII.) Inhibition of 161P2F10B Protein Function        -   XII.A.) Inhibition of 161P2F10B With Intracellular            Antibodies        -   XII.B.) Inhibition of 161P2F10B with Recombinant Proteins        -   XII.C.) Inhibition of 161P2F10B Transcription or Translation        -   XII.D.) General Considerations for Therapeutic Strategies    -   XIII.) Identification, Characterization and Use of Modulators of        161P2F10b    -   XIV.) KITS/Articles of Manufacture

I.) Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art, such as, forexample, the widely utilized molecular cloning methodologies describedin Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Asappropriate, procedures involving the use of commercially available kitsand reagents are generally carried out in accordance with manufacturerdefined protocols and/or parameters unless otherwise noted.

The terms “advanced prostate cancer”, “locally advanced prostatecancer”, “advanced disease” and “locally advanced disease” mean prostatecancers that have extended through the prostate capsule, and are meantto include stage C disease under the American Urological Association(AUA) system, stage C1-C2 disease under the Whitmore-Jewett system, andstage T3-T4 and N+ disease under the TNM (tumor, node, metastasis)system. In general, surgery is not recommended for patients with locallyadvanced disease, and these patients have substantially less favorableoutcomes compared to patients having clinically localized(organ-confined) prostate cancer. Locally advanced disease is clinicallyidentified by palpable evidence of induration beyond the lateral borderof the prostate, or asymmetry or induration above the prostate base.Locally advanced prostate cancer is presently diagnosed pathologicallyfollowing radical prostatectomy if the tumor invades or penetrates theprostatic capsule, extends into the surgical margin, or invades theseminal vesicles.

“Altering the native glycosylation pattern” is intended for purposesherein to mean deleting one or more carbohydrate moieties found innative sequence 161P2F10B (either by removing the underlyingglycosylation site or by deleting the glycosylation by chemical and/orenzymatic means), and/or adding one or more glycosylation sites that arenot present in the native sequence 161P2F10B. In addition, the phraseincludes qualitative changes in the glycosylation of the nativeproteins, involving a change in the nature and proportions of thevarious carbohydrate moieties present.

The term “analog” refers to a molecule which is structurally similar orshares similar or corresponding attributes with another molecule (e.g. a161P2F10B-related protein). For example, an analog of a 161P2F10Bprotein can be specifically bound by an antibody or T cell thatspecifically binds to 161P2F10B.

The term “antibody” is used in the broadest sense. Therefore, an“antibody” can be naturally occurring or man-made such as monoclonalantibodies produced by conventional hybridoma technology. Anti-161P2F10Bantibodies comprise monoclonal and polyclonal antibodies as well asfragments containing the antigen-binding domain and/or one or morecomplementarity determining regions of these antibodies.

An “antibody fragment” is defined as at least a portion of the variableregion of the immunoglobulin molecule that binds to its target, i.e.,the antigen-binding region. In one embodiment it specifically coverssingle anti-161P2F10B antibodies and clones thereof (including agonist,antagonist and neutralizing antibodies) and anti-161P2F10B antibodycompositions with polyepitopic specificity.

The term “codon optimized sequences” refers to nucleotide sequences thathave been optimized for a particular host species by replacing anycodons having a usage frequency of less than about 20%. Nucleotidesequences that have been optimized for expression in a given hostspecies by elimination of spurious polyadenylation sequences,elimination of exon/intron splicing signals, elimination oftransposon-like repeats and/or optimization of GC content in addition tocodon optimization are referred to herein as an “expression enhancedsequences.”

A “combinatorial library” is a collection of diverse chemical compoundsgenerated by either chemical synthesis or biological synthesis bycombining a number of chemical “building blocks” such as reagents. Forexample, a linear combinatorial chemical library, such as a polypeptide(e.g., mutein) library, is formed by combining a set of chemicalbuilding blocks called amino acids in every possible way for a givencompound length (i.e., the number of amino acids in a polypeptidecompound). Numerous chemical compounds are synthesized through suchcombinatorial mixing of chemical building blocks (Gallop et al., J. Med.Chem. 37(9): 1233-1251 (1994)).

Preparation and screening of combinatorial libraries is well known tothose of skill in the art. Such combinatorial chemical librariesinclude, but are not limited to, peptide libraries (see, e.g., U.S. Pat.No. 5,010,175, Furka, Pept. Prot. Res. 37:487-493 (1991), Houghton etal., Nature, 354:84-88 (1991)), peptoids (PCT Publication No WO91/19735), encoded peptides (PCT Publication WO 93/20242), randombio-oligomers (PCT Publication WO 92/00091), benzodiazepines (U.S. Pat.No. 5,288,514), diversomers such as hydantoins, benzodiazepines anddipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913(1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc.114:6568 (1992)), nonpeptidal peptidomimetics with a Beta-D-Glucosescaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218(1992)), analogous organic syntheses of small compound libraries (Chenet al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbarnates (Cho, etal., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell etal., J. Org. Chem. 59:658 (1994)). See, generally, Gordon et al., J.Med. Chem. 37:1385 (1994), nucleic acid libraries (see, e.g.,Stratagene, Corp.), peptide nucleic acid libraries (see, e.g., U.S. Pat.No. 5,539,083), antibody libraries (see, e.g., Vaughn et al., NatureBiotechnology 14(3): 309-314 (1996), and PCT/US96/10287), carbohydratelibraries (see, e.g., Liang et al., Science 274:1520-1522 (1996), andU.S. Pat. No. 5,593,853), and small organic molecule libraries (see,e.g., benzodiazepines, Baum, C&EN, January 18, page 33 (1993);isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones andmetathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos.5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337;benzodiazepines, U.S. Pat. No. 5,288,514; and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 NIPS, 390 NIPS, Advanced Chem Tech, LouisvilleKy.; Symphony, Rainin, Woburn, Mass.; 433A, Applied Biosystems, FosterCity, Calif.; 9050, Plus, Millipore, Bedford, Mass.). A number ofwell-known robotic systems have also been developed for solution phasechemistries. These systems include automated workstations such as theautomated synthesis apparatus developed by Takeda Chemical Industries,LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms(Zymate H, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard,Palo Alto, Calif.), which mimic the manual synthetic operationsperformed by a chemist. Any of the above devices are suitable for usewith the present invention. The nature and implementation ofmodifications to these devices (if any) so that they can operate asdiscussed herein will be apparent to persons skilled in the relevantart. In addition, numerous combinatorial libraries are themselvescommercially available (see, e.g., ComGenex, Princeton, N.J.; Asinex,Moscow, RU; Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd, Moscow, RU; 3DPharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md.; etc.).

The term “cytotoxic agent” refers to a substance that inhibits orprevents the expression activity of cells, function of cells and/orcauses destruction of cells. The term is intended to include radioactiveisotopes chemotherapeutic agents, and toxins such as small moleculetoxins or enzymatically active toxins of bacterial, fungal, plant oranimal origin, including fragments and/or variants thereof. Examples ofcytotoxic agents include, but are not limited to auristatins,auromycins, maytansinoids, yttrium, bismuth, ricin, ricin A-chain,combrestatin, duocarmycins, dolostatins, doxorubicin, daunorubicin,taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracindione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40,abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin,mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin,calicheamicin, Saponaria officinalis inhibitor, and glucocorticoid andother chemotherapeutic agents, as well as radioisotopes such as At211,I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32 and radioactiveisotopes of Lu including Lu177. Antibodies may also be conjugated to ananti-cancer pro-drug activating enzyme capable of converting thepro-drug to its active form.

The “gene product” is sometimes referred to herein as a protein or mRNA.For example, a “gene product of the invention” is sometimes referred toherein as a “cancer amino acid sequence”, “cancer protein”, “protein ofa cancer listed in Table I”, a “cancer mRNA”, “mRNA of a cancer listedin Table I”, etc. In one embodiment, the cancer protein is encoded by anucleic acid of FIG. 2. The cancer protein can be a fragment, oralternatively, be the full-length protein to the fragment encoded by thenucleic acids of FIG. 2. In one embodiment, a cancer amino acid sequenceis used to determine sequence identity or similarity. In anotherembodiment, the sequences are naturally occurring allelic variants of aprotein encoded by a nucleic acid of FIG. 2. In another embodiment, thesequences are sequence variants as further described herein.

“High throughput screening” assays for the presence, absence,quantification, or other properties of particular nucleic acids orprotein products are well known to those of skill in the art. Similarly,binding assays and reporter gene assays are similarly well known. Thus,e.g., U.S. Pat. No. 5,559,410 discloses high throughput screeningmethods for proteins; U.S. Pat. No. 5,585,639 discloses high throughputscreening methods for nucleic acid binding (i.e., in arrays); while U.S.Pat. Nos. 5,576,220 and 5,541,061 disclose high throughput methods ofscreening for ligand/antibody binding.

In addition, high throughput screening systems are commerciallyavailable (see, e.g., Amersham Biosciences, Piscataway, N.J.; ZymarkCorp., Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio; BeckmanInstruments, Inc. Fullerton, Calif.; Precision Systems, Inc., Natick,Mass.; etc.). These systems typically automate entire procedures,including all sample and reagent pipetting, liquid dispensing, timedincubations, and final readings of the microplate in detector(s)appropriate for the assay. These configurable systems provide highthroughput and rapid start up as well as a high degree of flexibilityand customization. The manufacturers of such systems provide detailedprotocols for various high throughput systems. Thus, e.g., Zymark Corp.provides technical bulletins describing screening systems for detectingthe modulation of gene transcription, ligand binding, and the like.

The term “homolog” refers to a molecule which exhibits homology toanother molecule, by for example, having sequences of chemical residuesthat are the same or similar at corresponding positions.

“Human Leukocyte Antigen” or “HLA” is a human class I or class II MajorHistocompatibility Complex (MHC) protein (see, e.g., Stites, et al.,Immunology, 8th Ed., Lange Publishing, Los Altos, Calif. (1994).

The terms “hybridize”, “hybridizing”, “hybridizes” and the like, used inthe context of polynucleotides, are meant to refer to conventionalhybridization conditions, preferably such as hybridization in 50%formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperatures forhybridization are above 37 degrees C. and temperatures for washing in0.1×SSC/0.1% SDS are above 55 degrees C.

The phrases “isolated” or “biologically pure” refer to material which issubstantially or essentially free from components which normallyaccompany the material as it is found in its native state. Thus,isolated peptides in accordance with the invention preferably do notcontain materials normally associated with the peptides in their in situenvironment. For example, a polynucleotide is said to be “isolated” whenit is substantially separated from contaminant polynucleotides thatcorrespond or are complementary to genes other than the 161P2F10B genesor that encode polypeptides other than 161P2F10B gene product orfragments thereof. A skilled artisan can readily employ nucleic acidisolation procedures to obtain an isolated 161P2F10B polynucleotide. Aprotein is said to be “isolated,” for example, when physical, mechanicalor chemical methods are employed to remove the 161P2F10B proteins fromcellular constituents that are normally associated with the protein. Askilled artisan can readily employ standard purification methods toobtain an isolated 161P2F10B protein. Alternatively, an isolated proteincan be prepared by chemical means.

The term “mammal” refers to any organism classified as a mammal,including mice, rats, rabbits, dogs, cats, cows, horses and humans. Inone embodiment of the invention, the mammal is a mouse. In anotherembodiment of the invention, the mammal is a human.

The terms “metastatic prostate cancer” and “metastatic disease” meanprostate cancers that have spread to regional lymph nodes or to distantsites, and are meant to include stage D disease under the AUA system andstage TxNxM+ under the TNM system. As is the case with locally advancedprostate cancer, surgery is generally not indicated for patients withmetastatic disease, and hormonal (androgen ablation) therapy is apreferred treatment modality. Patients with metastatic prostate cancereventually develop an androgen-refractory state within 12 to 18 monthsof treatment initiation. Approximately half of these androgen-refractorypatients die within 6 months after developing that status. The mostcommon site for prostate cancer metastasis is bone. Prostate cancer bonemetastases are often osteoblastic rather than osteolytic (i.e.,resulting in net bone formation). Bone metastases are found mostfrequently in the spine, followed by the femur, pelvis, rib cage, skulland humerus. Other common sites for metastasis include lymph nodes,lung, liver and brain. Metastatic prostate cancer is typically diagnosedby open or laparoscopic pelvic lymphadenectomy, whole body radionuclidescans, skeletal radiography, and/or bone lesion biopsy.

The term “modulator” or “test compound” or “drug candidate” orgrammatical equivalents as used herein describe any molecule, e.g.,protein, oligopeptide, small organic molecule, polysaccharide,polynucleotide, etc., to be tested for the capacity to directly orindirectly alter the cancer phenotype or the expression of a cancersequence, e.g., a nucleic acid or protein sequences, or effects ofcancer sequences (e.g., signaling, gene expression, protein interaction,etc.) In one aspect, a modulator will neutralize the effect of a cancerprotein of the invention. By “neutralize” is meant that an activity of aprotein is inhibited or blocked, along with the consequent effect on thecell. In another aspect, a modulator will neutralize the effect of agene, and its corresponding protein, of the invention by normalizinglevels of said protein. In preferred embodiments, modulators alterexpression profiles, or expression profile nucleic acids or proteinsprovided herein, or downstream effector pathways. In one embodiment, themodulator suppresses a cancer phenotype, e.g. to a normal tissuefingerprint. In another embodiment, a modulator induced a cancerphenotype. Generally, a plurality of assay mixtures is run in parallelwith different agent concentrations to obtain a differential response tothe various concentrations. Typically, one of these concentrationsserves as a negative control, i.e., at zero concentration or below thelevel of detection.

Modulators, drug candidates or test compounds encompass numerouschemical classes, though typically they are organic molecules,preferably small organic compounds having a molecular weight of morethan 100 and less than about 2,500 Daltons. Preferred small moleculesare less than 2000, or less than 1500 or less than 1000 or less than 500D. Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Modulators also comprise biomolecules such aspeptides, saccharides, fatty acids, steroids, purines, pyrimidines,derivatives, structural analogs or combinations thereof. Particularlypreferred are peptides. One class of modulators are peptides, forexample of from about five to about 35 amino acids, with from about fiveto about 20 amino acids being preferred, and from about 7 to about 15being particularly preferred. Preferably, the cancer modulatory proteinis soluble, includes a non-transmembrane region, and/or, has anN-terminal Cys to aid in solubility. In one embodiment, the C-terminusof the fragment is kept as a free acid and the N-terminus is a freeamine to aid in coupling, i.e., to cysteine. In one embodiment, a cancerprotein of the invention is conjugated to an immunogenic agent asdiscussed herein. In one embodiment, the cancer protein is conjugated toBSA. The peptides of the invention, e.g., of preferred lengths, can belinked to each other or to other amino acids to create a longerpeptide/protein. The modulatory peptides can be digests of naturallyoccurring proteins as is outlined above, random peptides, or “biased”random peptides. In a preferred embodiment, peptide/protein-basedmodulators are antibodies, and fragments thereof, as defined herein.

Modulators of cancer can also be nucleic acids. Nucleic acid modulatingagents can be naturally occurring nucleic acids, random nucleic acids,or “biased” random nucleic acids. For example, digests of prokaryotic oreukaryotic genomes can be used in an approach analogous to that outlinedabove for proteins.

The term “monoclonal antibody” refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the antibodiescomprising the population are identical except for possible naturallyoccurring mutations that are present in minor amounts.

A “motif”, as in biological motif of a 161P2F10B-related protein, refersto any pattern of amino acids forming part of the primary sequence of aprotein, that is associated with a particular function (e.g.protein-protein interaction, protein-DNA interaction, etc) ormodification (e.g. that is phosphorylated, glycosylated or amidated), orlocalization (e.g. secretory sequence, nuclear localization sequence,etc.) or a sequence that is correlated with being immunogenic, eitherhumorally or cellularly. A motif can be either contiguous or capable ofbeing aligned to certain positions that are generally correlated with acertain function or property. In the context of HLA motifs, “motif”refers to the pattern of residues in a peptide of defined length,usually a peptide of from about 8 to about 13 amino acids for a class IHLA motif and from about 6 to about 25 amino acids for a class II HLAmotif, which is recognized by a particular HLA molecule. Peptide motifsfor HLA binding are typically different for each protein encoded by eachhuman HLA allele and differ in the pattern of the primary and secondaryanchor residues.

A “pharmaceutical excipient” comprises a material such as an adjuvant, acarrier, pH-adjusting and buffering agents, tonicity adjusting agents,wetting agents, preservative, and the like.

“Pharmaceutically acceptable” refers to a non-toxic, inert, and/orcomposition that is physiologically compatible with humans or othermammals.

The term “polynucleotide” means a polymeric form of nucleotides of atleast 10 bases or base pairs in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide, and ismeant to include single and double stranded forms of DNA and/or RNA. Inthe art, this term if often used interchangeably with “oligonucleotide”.A polynucleotide can comprise a nucleotide sequence disclosed hereinwherein thymidine (T), as shown for example in FIG. 2, can also beuracil (U); this definition pertains to the differences between thechemical structures of DNA and RNA, in particular the observation thatone of the four major bases in RNA is uracil (U) instead of thymidine(T).

The term “polypeptide” means a polymer of at least about 4, 5, 6, 7, or8 amino acids. Throughout the specification, standard three letter orsingle letter designations for amino acids are used. In the art, thisterm is often used interchangeably with “peptide” or “protein”.

An HLA “primary anchor residue” is an amino acid at a specific positionalong a peptide sequence which is understood to provide a contact pointbetween the immunogenic peptide and the HLA molecule. One to three,usually two, primary anchor residues within a peptide of defined lengthgenerally defines a “motif” for an immunogenic peptide. These residuesare understood to fit in close contact with peptide binding groove of anHLA molecule, with their side chains buried in specific pockets of thebinding groove. In one embodiment, for example, the primary anchorresidues for an HLA class I molecule are located at position 2 (from theamino terminal position) and at the carboxyl terminal position of a 8,9, 10, 11, or 12 residue peptide epitope in accordance with theinvention. Alternatively, in another embodiment, the primary anchorresidues of a peptide binds an HLA class II molecule are spaced relativeto each other, rather than to the termini of a peptide, where thepeptide is generally of at least 9 amino acids in length. The primaryanchor positions for each motif and supermotif are set forth in TableIV. For example, analog peptides can be created by altering the presenceor absence of particular residues in the primary and/or secondary anchorpositions shown in Table IV. Such analogs are used to modulate thebinding affinity and/or population coverage of a peptide comprising aparticular HLA motif or supermotif.

“Radioisotopes” include, but are not limited to the following(non-limiting exemplary uses are also set forth below, particularlyexamples of medical isotopes):

Isotopes Description of use Actinium-225 See Thorium-229 (Th-229)(AC-225) Actinium-227 Parent of Radium-223 (Ra-223) which is an alphaemitter used to treat metastases in (AC-227) the skeleton resulting fromcancer (i.e., breast and prostate cancers), and cancerradioimmunotherapy Bismuth-212 See Thorium-228 (Th-228) (Bi-212)Bismuth-213 See Thorium-229 (Th-229) (Bi-213) Cadmium-109 Cancerdetection (Cd-109) Cobalt-60 Radiation source for radiotherapy ofcancer, for food irradiators, and for sterilization of (Co-60) medicalsupplies Copper-64 A positron emitter used for cancer therapy and SPECTimaging (Cu-64) Copper-67 Beta/gamma emitter used in cancerradioimmunotherapy and diagnostic studies (i.e., breast (Cu-67) andcolon cancers, and lymphoma) Dysprosium-166 Cancer radioimmunotherapy(Dy-166) Erbium-169 Rheumatoid arthritis treatment, particularly for thesmall joints associated with fingers and (Er-169) toes Europium-152Radiation source for food irradiation and for sterilization of medicalsupplies (Eu-152) Europium-154 Radiation source for food irradiation andfor sterilization of medical supplies (Eu-154) Gadolinium-153Osteoporosis detection and nuclear medical quality assurance devices(Gd-153) Gold-198 Implant and intracavity therapy of ovarian, prostate,and brain cancers (Au-198) Holmium-166 Multiple myeloma treatment intargeted skeletal therapy, cancer radioimmunotherapy, bone (Ho-166)marrow ablation, and rheumatoid arthritis treatment Iodine-125Osteoporosis detection, diagnostic imaging, tracer drugs, brain cancertreatment, (I-125) radiolabeling, tumor imaging, mapping of receptors inthe brain, interstitial radiation therapy, brachytherapy for treatmentof prostate cancer, determination of glomerular filtration rate (GFR),determination of plasma volume, detection of deep vein thrombosis of thelegs Iodine-131 Thyroid function evaluation, thyroid disease detection,treatment of thyroid cancer as well as (I-131) other non-malignantthyroid diseases (i.e., Graves disease, goiters, and hyperthyroidism),treatment of leukemia, lymphoma, and other forms of cancer (e.g., breastcancer) using radioimmunotherapy Iridium-192 Brachytherapy, brain andspinal cord tumor treatment, treatment of blocked arteries (i.e.,(Ir-192) arteriosclerosis and restenosis), and implants for breast andprostate tumors Lutetium-177 Cancer radioimmunotherapy and treatment ofblocked arteries (i.e., arteriosclerosis and (Lu-177) restenosis)Molybdenum-99 Parent of Technetium-99m (Tc-99m) which is used forimaging the brain, liver, lungs, heart, (Mo-99) and other organs.Currently, Tc-99m is the most widely used radioisotope used fordiagnostic imaging of various cancers and diseases involving the brain,heart, liver, lungs; also used in detection of deep vein thrombosis ofthe legs Osmium-194 Cancer radioimmunotherapy (Os-194) Palladium-103Prostate cancer treatment (Pd-103) Platinum-195m Studies onbiodistribution and metabolism of cisplatin, a chemotherapeutic drug(Pt-195m) Phosphorus-32 Polycythemia rubra vera (blood cell disease) andleukemia treatment, bone cancer (P-32) diagnosis/treatment; colon,pancreatic, and liver cancer treatment; radiolabeling nucleic acids forin vitro research, diagnosis of superficial tumors, treatment of blockedarteries (i.e., arteriosclerosis and restenosis), and intracavitytherapy Phosphorus-33 Leukemia treatment, bone diseasediagnosis/treatment, radiolabeling, and treatment of (P-33) blockedarteries (i.e., arteriosclerosis and restenosis) Radium-223 SeeActinium-227 (Ac-227) (Ra-223) Rhenium-186 Bone cancer pain relief,rheumatoid arthritis treatment, and diagnosis and treatment of (Re-186)lymphoma and bone, breast, colon, and liver cancers usingradioimmunotherapy Rhenium-188 Cancer diagnosis and treatment usingradioimmunotherapy, bone cancer pain relief, (Re-188) treatment ofrheumatoid arthritis, and treatment of prostate cancer Rhodium-105Cancer radioimmunotherapy (Rh-105) Samarium-145 Ocular cancer treatment(Sm-145) Samarium-153 Cancer radioimmunotherapy and bone cancer painrelief (Sm-153) Scandium-47 Cancer radioimmunotherapy and bone cancerpain relief (Sc-47) Selenium-75 Radiotracer used in brain studies,imaging of adrenal cortex by gamma-scintigraphy, lateral (Se-75)locations of steroid secreting tumors, pancreatic scanning, detection ofhyperactive parathyroid glands, measure rate of bile acid loss from theendogenous pool Strontium-85 Bone cancer detection and brain scans(Sr-85) Strontium-89 Bone cancer pain relief, multiple myelomatreatment, and osteoblastic therapy (Sr-89) Technetium-99m SeeMolybdenum-99 (Mo-99) (Tc-99m) Thorium-228 Parent of Bismuth-212(Bi-212) which is an alpha emitter used in cancer radioimmunotherapy(Th-228) Thorium-229 Parent of Actinium-225 (Ac-225) and grandparent ofBismuth-213 (Bi-213) which are alpha (Th-229) emitters used in cancerradioimmunotherapy Thulium-170 Gamma source for blood irradiators,energy source for implanted medical devices (Tm-170) Tin-117m Cancerimmunotherapy and bone cancer pain relief (Sn-117m) Tungsten-188 Parentfor Rhenium-188 (Re-188) which is used for cancer diagnostics/treatment,bone (W-188) cancer pain relief, rheumatoid arthritis treatment, andtreatment of blocked arteries (i.e., arteriosclerosis and restenosis)Xenon-127 Neuroimaging of brain disorders, high resolution SPECTstudies, pulmonary function tests, (Xe-127) and cerebral blood flowstudies Ytterbium-175 Cancer radioimmunotherapy (Yb-175) Yttrium-90Microseeds obtained from irradiating Yttrium-89 (Y-89) for liver cancertreatment (Y-90) Yttrium-91 A gamma-emitting label for Yttrium-90 (Y-90)which is used for cancer radioimmunotherapy (Y-91) (i.e., lymphoma,breast, colon, kidney, lung, ovarian, prostate, pancreatic, andinoperable liver cancers)

By “randomized” or grammatical equivalents as herein applied to nucleicacids and proteins is meant that each nucleic acid and peptide consistsof essentially random nucleotides and amino acids, respectively. Theserandom peptides (or nucleic acids, discussed herein) can incorporate anynucleotide or amino acid at any position. The synthetic process can bedesigned to generate randomized proteins or nucleic acids, to allow theformation of all or most of the possible combinations over the length ofthe sequence, thus forming a library of randomized candidate bioactiveproteinaceous agents.

In one embodiment, a library is “fully randomized,” with no sequencepreferences or constants at any position. In another embodiment, thelibrary is a “biased random” library. That is, some positions within thesequence either are held constant, or are selected from a limited numberof possibilities. For example, the nucleotides or amino acid residuesare randomized within a defined class, e.g., of hydrophobic amino acids,hydrophilic residues, sterically biased (either small or large)residues, towards the creation of nucleic acid binding domains, thecreation of cysteines, for cross-linking, prolines for SH-3 domains,serines, threonines, tyrosines or histidines for phosphorylation sites,etc., or to purines, etc.

A “recombinant” DNA or RNA molecule is a DNA or RNA molecule that hasbeen subjected to molecular manipulation in vitro.

Non-limiting examples of small molecules include compounds that bind orinteract with 161P2F10B, ligands including hormones, neuropeptides,chemokines, odorants, phospholipids, and functional equivalents thereofthat bind and preferably inhibit 161P2F10B protein function. Suchnon-limiting small molecules preferably have a molecular weight of lessthan about 10 kDa, more preferably below about 9, about 8, about 7,about 6, about 5 or about 4 kDa. In certain embodiments, small moleculesphysically associate with, or bind, 161P2F10B protein; are not found innaturally occurring metabolic pathways; and/or are more soluble inaqueous than non-aqueous solutions

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured nucleic acidsequences to reanneal when complementary strands are present in anenvironment below their melting temperature. The higher the degree ofdesired homology between the probe and hybridizable sequence, the higherthe relative temperature that can be used. As a result, it follows thathigher relative temperatures would tend to make the reaction conditionsmore stringent, while lower temperatures less so. For additional detailsand explanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, are identified by, but not limited to, those that: (1) employlow ionic strength and high temperature for washing, for example 0.015 Msodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at50° C.; (2) employ during hybridization a denaturing agent, such asformamide, for example, 50% (v/v) formamide with 0.1% bovine serumalbumin/0 1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS,and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC(sodium chloride/sodium. citrate) and 50% formamide at 55° C., followedby a high-stringency wash consisting of 0.1×SSC containing EDTA at 55°C. “Moderately stringent conditions” are described by, but not limitedto, those in Sambrook et al., Molecular Cloning: A Laboratory Manual,New York: Cold Spring Harbor Press, 1989, and include the use of washingsolution and hybridization conditions (e.g., temperature, ionic strengthand % SDS) less stringent than those described above. An example ofmoderately stringent conditions is overnight incubation at 37° C. in asolution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10%dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA,followed by washing the filters in 1×SSC at about 37-50° C. The skilledartisan will recognize how to adjust the temperature, ionic strength,etc. as necessary to accommodate factors such as probe length and thelike.

An HLA “supermotif” is a peptide binding specificity shared by HLAmolecules encoded by two or more HLA alleles. Overall phenotypicfrequencies of HLA-supertypes in different ethnic populations are setforth in Table IV (F). The non-limiting constituents of varioussupertypes are as follows:

-   -   A2: A*0201, A*0202, A*0203, A*0204, A* 0205, A*0206, A*6802,        A*6901, A*0207    -   A3: A3, A11, A31, A*3301, A*6801, A*0301, A*1101, A*3101    -   B7: B7, B*3501-03, B*51, B*5301, B*5401, B*5501, B*5502, B*5601,        B*6701, B*7801, B*0702, B*5101, B*5602    -   B44: B*3701, B*4402, B*4403, B*60 (B*4001), B61 (B*4006)    -   A1: A*0102, A*2604, A*3601, A*4301, A*8001    -   A24: A*24, A*30, A*2403, A*2404, A*3002, A*3003    -   B27: B*1401-02, B*1503, B*1509, B*1510, B*1518, B*3801-02,        B*3901, B*3902, B*3903-04, B*4801-02, B*7301, B*2701-08    -   B58: B*1516, B*1517, B*5701, B*5702, B58    -   B62: B*4601, B52, B*1501 (B62), B*1502 (B75), B*1513 (B77)

Calculated population coverage afforded by different HLA-supertypecombinations are set forth in Table IV (G).

As used herein “to treat” or “therapeutic” and grammatically relatedterms, refer to any improvement of any consequence of disease, such asprolonged survival, less morbidity, and/or a lessening of side effectswhich are the byproducts of an alternative therapeutic modality; fulleradication of disease is not required.

A “transgenic animal” (e.g., a mouse or rat) is an animal having cellsthat contain a transgene, which transgene was introduced into the animalor an ancestor of the animal at a prenatal, e.g., an embryonic stage. A“transgene” is a DNA that is integrated into the genome of a cell fromwhich a transgenic animal develops.

As used herein, an HLA or cellular immune response “vaccine” is acomposition that contains or encodes one or more peptides of theinvention. There are numerous embodiments of such vaccines, such as acocktail of one or more individual peptides; one or more peptides of theinvention comprised by a polyepitopic peptide; or nucleic acids thatencode such individual peptides or polypeptides, e.g., a minigene thatencodes a polyepitopic peptide. The “one or more peptides” can includeany whole unit integer from 1-150 or more, e.g., at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more peptides ofthe invention. The peptides or polypeptides can optionally be modified,such as by lipidation, addition of targeting or other sequences. HLAclass I peptides of the invention can be admixed with, or linked to, HLAclass II peptides, to facilitate activation of both cytotoxic Tlymphocytes and helper T lymphocytes. HLA vaccines can also comprisepeptide-pulsed antigen presenting cells, e.g., dendritic cells.

The term “variant” refers to a molecule that exhibits a variation from adescribed type or norm, such as a protein that has one or more differentamino acid residues in the corresponding position(s) of a specificallydescribed protein (e.g. the 161P2F10B protein shown in FIG. 2 or FIG. 3.An analog is an example of a variant protein. Splice isoforms and singlenucleotides polymorphisms (SNPs) are further examples of variants.

The “161P2F10B-related proteins” of the invention include thosespecifically identified herein, as well as allelic variants,conservative substitution variants, analogs and homologs that can beisolated/generated and characterized without undue experimentationfollowing the methods outlined herein or readily available in the art.Fusion proteins that combine parts of different 161P2F10B proteins orfragments thereof, as well as fusion proteins of a 161P2F10B protein anda heterologous polypeptide are also included. Such 161P2F10B proteinsare collectively referred to as the 161P2F10B-related proteins, theproteins of the invention, or 161P2F10B. The term “161P2F10B-relatedprotein” refers to a polypeptide fragment or a 161P2F10B proteinsequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, or more than 25 amino acids; or, at least 30,35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120,125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190,195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500,525, 550, 575, 600, 625, 650, or 664 or more amino acids.

II.) 161P2F10B Polynucleotides

One aspect of the invention provides polynucleotides corresponding orcomplementary to all or part of a 161P2F10B gene, mRNA, and/or codingsequence, preferably in isolated form, including polynucleotidesencoding a 161P2F10B-related protein and fragments thereof, DNA, RNA,DNA/RNA hybrid, and related molecules, polynucleotides oroligonucleotides complementary to a 161P2F10B gene or mRNA sequence or apart thereof, and polynucleotides or oligonucleotides that hybridize toa 161P2F10B gene, mRNA, or to a 161P2F10B encoding polynucleotide(collectively, “161P2F10B polynucleotides”). In all instances whenreferred to in this section, T can also be U in FIG. 2.

Embodiments of a 161P2F10B polynucleotide include: a 161P2F10Bpolynucleotide having the sequence shown in FIG. 2, the nucleotidesequence of 161P2F10B as shown in FIG. 2 wherein T is U; at least 10contiguous nucleotides of a polynucleotide having the sequence as shownin FIG. 2; or, at least 10 contiguous nucleotides of a polynucleotidehaving the sequence as shown in FIG. 2 where T is U. For example,embodiments of 161P2F10B nucleotides comprise, without limitation:

(I) a polynucleotide comprising, consisting essentially of, orconsisting of a sequence as shown in FIG. 2, wherein T can also be U;

(II) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2A, from nucleotide residuenumber 44 through nucleotide residue number 2671, including the stopcodon, wherein T can also be U;

(III) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2B, from nucleotide residuenumber 44 through nucleotide residue number 2671, including the stopcodon, wherein T can also be U;

(IV) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2C, from nucleotide residuenumber 44 through nucleotide residue number 2671, including the a stopcodon, wherein T can also be U;

(V) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2D, from nucleotide residuenumber 44 through nucleotide residue number 2671, including the stopcodon, wherein T can also be U;

(VI) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2E, from nucleotide residuenumber 44 through nucleotide residue number 2671, including the stopcodon, wherein T can also be U;

(VII) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2F, from nucleotide residuenumber 84 through nucleotide residue number 2711, including the stopcodon, wherein T can also be U;

(VIII) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2G, from nucleotide residuenumber 276 through nucleotide residue number 2801, including the stopcodon, wherein T can also be U;

(IX) a polynucleotide that encodes a 161P2F10B-related protein that isat least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homologous to anentire amino acid sequence shown in FIG. 2A-G;

(X) a polynucleotide that encodes a 161P2F10B-related protein that is atleast 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to anentire amino acid sequence shown in FIG. 2A-G;

(XI) a polynucleotide that encodes at least one peptide set forth inTables VIII-XXI and XXII-XLIX;

(XII) a polynucleotide that encodes a peptide region of at least 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG.3A-D in any whole number increment up to 875 that includes at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acidposition(s) having a value greater than 0.5 in the Hydrophilicityprofile of FIG. 5;

(XIII) a polynucleotide that encodes a peptide region of at least 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG.3A-D in any whole number increment up to 875 that includes 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s)having a value less than 0.5 in the Hydropathicity profile of FIG. 6;

(XIV) a polynucleotide that encodes a peptide region of at least 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG.3A-D in any whole number increment up to 875 that includes 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s)having a value greater than 0.5 in the Percent Accessible Residuesprofile of FIG. 7;

(XV) a polynucleotide that encodes a peptide region of at least 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG.3A-DF in any whole number increment up to 875 that includes 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s)having a value greater than 0.5 in the Average Flexibility profile ofFIG. 8;

(XVI) a polynucleotide that encodes a peptide region of at least 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG.3A-D in any whole number increment up to 875 that includes 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s)having a value greater than 0.5 in the Beta-turn profile of FIG. 9;

(XVII) a polynucleotide that encodes a peptide region of at least 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG.3E in any whole number increment up to 841 that includes 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) havinga value greater than 0.5 in the Hydrophilicity profile of FIG. 5;

(XVIII) a polynucleotide that encodes a peptide region of at least 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG.3E in any whole number increment up to 841 that includes 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) havinga value less than 0.5 in the Hydropathicity profile of FIG. 6;

(XIX) a polynucleotide that encodes a peptide region of at least 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG.3E in any whole number increment up to 841 that includes 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) havinga value greater than 0.5 in the Percent Accessible Residues profile ofFIG. 7;

(XX) a polynucleotide that encodes a peptide region of at least 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG.3E in any whole number increment up to 841 that includes 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) havinga value greater than 0.5 in the Average Flexibility profile of FIG. 8;

(XXI) a polynucleotide that encodes a peptide region of at least 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG.3E in any whole number increment up to 841 that includes 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) havinga value greater than 0.5 in the Beta-turn profile of FIG. 9;

(XXII) a polynucleotide that encodes monoclonal antibody or bindingregion thereof secreted by a hybridoma entitled X41(3)15 deposited withthe American Type Culture Collection (ATCC; 10801 University Blvd.,Manassas, Va. 20110-2209 USA) on 7 Nov. 2002 and assigned as PatentDeposit Designation NO. PTA-4791;

XXIII) a polynucleotide that encodes monoclonal antibody or bindingregion thereof secreted by a hybridoma entitled X41(3)29 deposited withthe American Type Culture Collection (ATCC; 10801 University Blvd.,Manassas, Va. 20110-2209 USA) on 7 Nov. 2002 and assigned as PatentDeposit Designation NO. PTA-4791;

(XXIV) a polynucleotide that encodes monoclonal antibody or bindingregion thereof secreted by a hybridoma entitled X41(3)37 deposited withthe American Type Culture Collection (ATCC; 10801 University Blvd.,Manassas, Va. 20110-2209 USA) on 7 Nov. 2002 and assigned as PatentDeposit Designation NO. PTA-4791;

(XXV) a polynucleotide that encodes monoclonal antibody or bindingregion thereof secreted by a hybridoma entitled X41(4)6 deposited withthe American Type Culture Collection (ATCC; 10801 University Blvd.,Manassas, Va. 20110-2209 USA) on 7 Nov. 2002 and assigned as PatentDeposit Designation NO. PTA-4794;

(XXVI) a polynucleotide that encodes monoclonal antibody or bindingregion thereof secreted by a hybridoma entitled X41(3)17 deposited withthe American Type Culture Collection (ATCC; 10801 University Blvd.,Manassas, Va. 20110-2209 USA) on 7 Nov. 2002 and assigned as PatentDeposit Designation NO. PTA-4792;

(XXVII) a polynucleotide that encodes monoclonal antibody or bindingregion thereof secreted by a hybridoma entitled X41(3)50 deposited withthe American Type Culture Collection (ATCC; 10801 University Blvd.,Manassas, Va. 20110-2209 USA) on 7 Nov. 2002 and assigned as PatentDeposit Designation NO. PTA-4793;

(XXVIII) a polynucleotide that is fully complementary to apolynucleotide of any one of (I)-(XXVII).

(XXIX) a peptide that is encoded by any of (I) to (XXVII); and

(XXX) a composition comprising a polynucleotide of any of (I)-(XXVII) orpeptide of (XXIX) together with a pharmaceutical excipient and/or in ahuman unit dose form.

(XXXI) a method of using a polynucleotide of any (I)-(XXVII) or peptideof (XXIX) or a composition of (XXX) in a method to modulate a cellexpressing 161P2F10b,

(XXXII) a method of using a polynucleotide of any (I)-(XXVII) or peptideof (XXIX) or a composition of (XXX) in a method to diagnose, prophylax,prognose, or treat an individual who bears a cell expressing 161P2F10b

(XXXIII) a method of using a polynucleotide of any (I)-(XXVII) orpeptide of (XXIX) or a composition of (XXX) in a method to diagnose,prophylax, prognose, or treat an individual who bears a cell expressing161P2F10b, said cell from a cancer of a tissue listed in Table I;

(XXXIV) a method of using a polynucleotide of any (I)-(XLII) or peptideof (XXIX) or a composition of (XXX) in a method to diagnose, prophylax,prognose, or treat a cancer;

(XXXV) a method of using a polynucleotide of any (I)-(XLII) or peptideof (XXIX) or a composition of (XXX) in a method to diagnose, prophylax,prognose, or treat a cancer of a tissue listed in Table I; and,

(XXXVI) a method of using a polynucleotide of any (I)-(XLII) or peptideof (XXIX) or a composition of (XXX) in a method to identify orcharacterize a modulator of a cell expressing 161P2F10b.

As used herein, a range is understood to disclose specifically all wholeunit positions thereof.

Typical embodiments of the invention disclosed herein include 161P2F10Bpolynucleotides that encode specific portions of 161P2F10B mRNAsequences (and those which are complementary to such sequences) such asthose that encode the proteins and/or fragments thereof, for example:

(a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165,170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375,400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725,750, 775, 800, 825, 850, 860, 870, 875 or more contiguous amino acids of161P2F10B variant 1; the maximal lengths relevant for other variantsare: variant 2, 875 amino acids; variant 3, 875 amino acids, variant 4,875 amino acids, and variant 7, 841 amino acids.

For example, representative embodiments of the invention disclosedherein include: polynucleotides and their encoded peptides themselvesencoding about amino acid 1 to about amino acid 10 of the 161P2F10Bprotein shown in FIG. 2 or FIG. 3, polynucleotides encoding about aminoacid 10 to about amino acid 20 of the 161P2F10B protein shown in FIG. 2or FIG. 3, polynucleotides encoding about amino acid 20 to about aminoacid 30 of the 161P2F10B protein shown in FIG. 2 or FIG. 3,polynucleotides encoding about amino acid 30 to about amino acid 40 ofthe 161P2F10B protein shown in FIG. 2 or FIG. 3, polynucleotidesencoding about amino acid 40 to about amino acid 50 of the 161P2F10Bprotein shown in FIG. 2 or FIG. 3, polynucleotides encoding about aminoacid 50 to about amino acid 60 of the 161P2F10B protein shown in FIG. 2or FIG. 3, polynucleotides encoding about amino acid 60 to about aminoacid 70 of the 161P2F10B protein shown in FIG. 2 or FIG. 3,polynucleotides encoding about amino acid 70 to about amino acid 80 ofthe 161P2F10B protein shown in FIG. 2 or FIG. 3, polynucleotidesencoding about amino acid 80 to about amino acid 90 of the 161P2F10Bprotein shown in FIG. 2 or FIG. 3, polynucleotides encoding about aminoacid 90 to about amino acid 100 of the 161P2F10B protein shown in FIG. 2or FIG. 3, in increments of about 10 amino acids, ending at the carboxylterminal amino acid set forth in FIG. 2 or FIG. 3. Accordingly,polynucleotides encoding portions of the amino acid sequence (of about10 amino acids), of amino acids, 100 through the carboxyl terminal aminoacid of the 161P2F10B protein are embodiments of the invention. Whereinit is understood that each particular amino acid position discloses thatposition plus or minus five amino acid residues.

Polynucleotides encoding relatively long portions of a 161P2F10B proteinare also within the scope of the invention. For example, polynucleotidesencoding from about amino acid 1 (or 20 or 30 or 40 etc.) to about aminoacid 20, (or 30, or 40 or 50 etc.) of the 161P2F10B protein “or variant”shown in FIG. 2 or FIG. 3 can be generated by a variety of techniqueswell known in the art. These polynucleotide fragments can include anyportion of the 161P2F10B sequence as shown in FIG. 2.

Additional illustrative embodiments of the invention disclosed hereininclude 161P2F10B polynucleotide fragments encoding one or more of thebiological motifs contained within a 161P2F10B protein “or variant”sequence, including one or more of the motif-bearing subsequences of a161P2F10B protein “or variant” set forth in Tables VIII-XXI andXXII-XLIX. In another embodiment, typical polynucleotide fragments ofthe invention encode one or more of the regions of 161P2F10B protein orvariant that exhibit homology to a known molecule. In another embodimentof the invention, typical polynucleotide fragments can encode one ormore of the 161P2F10B protein or variant N-glycosylation sites, cAMP andcGMP-dependent protein kinase phosphorylation sites, casein kinase IIphosphorylation sites or N-myristoylation site and amidation sites.

Note that to determine the starting position of any peptide set forth inTables VIII-XXI and Tables XXII to XLIX (collectively HLA PeptideTables) respective to its parental protein, e.g., variant 1, variant 2,etc., reference is made to three factors: the particular variant, thelength of the peptide in an HLA Peptide Table, and the Search Peptideslisted in Table LVII. Generally, a unique Search Peptide is used toobtain HLA peptides for a particular variant. The position of eachSearch Peptide relative to its respective parent molecule is listed inTable VII. Accordingly, if a Search Peptide begins at position “X”, onemust add the value “X minus 1” to each position in Tables VIII-XXI andTables XXII-IL to obtain the actual position of the HLA peptides intheir parental molecule. For example if a particular Search Peptidebegins at position 150 of its parental molecule, one must add 150−1,i.e., 149 to each HLA peptide amino acid position to calculate theposition of that amino acid in the parent molecule.

II.A.) Uses of 161P2F10B Polynucleotides

II.A.1.) Monitoring of Genetic Abnormalities

The polynucleotides of the preceding paragraphs have a number ofdifferent specific uses. The human 161P2F10B gene maps to thechromosomal location set forth in the Example entitled “ChromosomalMapping of 161P2F10B.” For example, because the 161P2F10B gene maps tothis chromosome, polynucleotides that encode different regions of the161P2F10B proteins are used to characterize cytogenetic abnormalities ofthis chromosomal locale, such as abnormalities that are identified asbeing associated with various cancers. In certain genes, a variety ofchromosomal abnormalities including rearrangements have been identifiedas frequent cytogenetic abnormalities in a number of different cancers(see e.g. Krajinovic et al., Mutat. Res. 382(3-4): 81-83 (1998);Johansson et al., Blood 86(10): 3905-3914 (1995) and Finger et al.,P.N.A.S. 85(23): 9158-9162 (1988)). Thus, polynucleotides encodingspecific regions of the 161P2F10B proteins provide new tools that can beused to delineate, with greater precision than previously possible,cytogenetic abnormalities in the chromosomal region that encodes161P2F10B that may contribute to the malignant phenotype. In thiscontext, these polynucleotides satisfy a need in the art for expandingthe sensitivity of chromosomal screening in order to identify moresubtle and less common chromosomal abnormalities (see e.g. Evans et al.,Am. J. Obstet. Gynecol 171(4): 1055-1057 (1994)).

Furthermore, as 161P2F10B was shown to be highly expressed in bladderand other cancers, 161P2F10B polynucleotides are used in methodsassessing the status of 161P2F10B gene products in normal versuscancerous tissues. Typically, polynucleotides that encode specificregions of the 161P2F10B proteins are used to assess the presence ofperturbations (such as deletions, insertions, point mutations, oralterations resulting in a loss of an antigen etc.) in specific regionsof the 161P2F10B gene, such as regions containing one or more motifs.Exemplary assays include both RT-PCR assays as well as single-strandconformation polymorphism (SSCP) analysis (see, e.g., Marrogi et al., J.Cutan. Pathol. 26(8): 369-378 (1999), both of which utilizepolynucleotides encoding specific regions of a protein to examine theseregions within the protein.

II.A.2.) Antisense Embodiments

Other specifically contemplated nucleic acid related embodiments of theinvention disclosed herein are genomic DNA, cDNAs, ribozymes, andantisense molecules, as well as nucleic acid molecules based on analternative backbone, or including alternative bases, whether derivedfrom natural sources or synthesized, and include molecules capable ofinhibiting the RNA or protein expression of 161P2F10B. For example,antisense molecules can be RNAs or other molecules, including peptidenucleic acids (PNAs) or non-nucleic acid molecules such asphosphorothioate derivatives that specifically bind DNA or RNA in a basepair-dependent manner A skilled artisan can readily obtain these classesof nucleic acid molecules using the 161P2F10B polynucleotides andpolynucleotide sequences disclosed herein.

Antisense technology entails the administration of exogenousoligonucleotides that bind to a target polynucleotide located within thecells. The term “antisense” refers to the fact that sucholigonucleotides are complementary to their intracellular targets, e.g.,161P2F10B. See for example, Jack Cohen, Oligodeoxynucleotides, AntisenseInhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5(1988). The 161P2F10B antisense oligonucleotides of the presentinvention include derivatives such as S-oligonucleotides(phosphorothioate derivatives or S-oligos, see, Jack Cohen, supra),which exhibit enhanced cancer cell growth inhibitory action. S-oligos(nucleoside phosphorothioates) are isoelectronic analogs of anoligonucleotide (O-oligo) in which a nonbridging oxygen atom of thephosphate group is replaced by a sulfur atom. The S-oligos of thepresent invention can be prepared by treatment of the correspondingO-oligos with 3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfurtransfer reagent. See, e.g., Iyer, R. P. et al., J. Org. Chem.55:4693-4698 (1990); and Iyer, R. P. et al., J. Am. Chem. Soc.112:1253-1254 (1990). Additional 161P2F10B antisense oligonucleotides ofthe present invention include morpholino antisense oligonucleotidesknown in the art (see, e.g., Partridge et al., 1996, Antisense & NucleicAcid Drug Development 6: 169-175).

The 161P2F10B antisense oligonucleotides of the present inventiontypically can be RNA or DNA that is complementary to and stablyhybridizes with the first 100 5′ codons or last 100 3′ codons of a161P2F10B genomic sequence or the corresponding mRNA. Absolutecomplementarity is not required, although high degrees ofcomplementarity are preferred. Use of an oligonucleotide complementaryto this region allows for the selective hybridization to 161P2F10B mRNAand not to mRNA specifying other regulatory subunits of protein kinase.In one embodiment, 161P2F10B antisense oligonucleotides of the presentinvention are 15 to 30-mer fragments of the antisense DNA molecule thathave a sequence that hybridizes to 161P2F10B mRNA. Optionally, 161P2F10Bantisense oligonucleotide is a 30-mer oligonucleotide that iscomplementary to a region in the first 10 5′ codons or last 10 3′ codonsof 161P2F10B. Alternatively, the antisense molecules are modified toemploy ribozymes in the inhibition of 161P2F10B expression, see, e.g.,L. A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).

II.A.3.) Primers and Primer Pairs

Further specific embodiments of these nucleotides of the inventioninclude primers and primer pairs, which allow the specific amplificationof polynucleotides of the invention or of any specific parts thereof,and probes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Probes can be labeledwith a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers are used todetect the presence of a 161P2F10B polynucleotide in a sample and as ameans for detecting a cell expressing a 161P2F10B protein.

Examples of such probes include polypeptides comprising all or part ofthe human 161P2F10B cDNA sequence shown in FIG. 2. Examples of primerpairs capable of specifically amplifying 161P2F10B mRNAs are alsodescribed in the Examples. As will be understood by the skilled artisan,a great many different primers and probes can be prepared based on thesequences provided herein and used effectively to amplify and/or detecta 161P2F10B mRNA.

The 161P2F10B polynucleotides of the invention are useful for a varietyof purposes, including but not limited to their use as probes andprimers for the amplification and/or detection of the 161P2F10B gene(s),mRNA(s), or fragments thereof; as reagents for the diagnosis and/orprognosis of prostate cancer and other cancers; as coding sequencescapable of directing the expression of 161P2F10B polypeptides; as toolsfor modulating or inhibiting the expression of the 161P2F10B gene(s)and/or translation of the 161P2F10B transcript(s); and as therapeuticagents.

The present invention includes the use of any probe as described hereinto identify and isolate a 161P2F10B or 161P2F10B related nucleic acidsequence from a naturally occurring source, such as humans or othermammals, as well as the isolated nucleic acid sequence per se, whichwould comprise all or most of the sequences found in the probe used.

II.A.4.) Isolation of 161P2F10B-Encoding Nucleic Acid Molecules

The 161P2F10B cDNA sequences described herein enable the isolation ofother polynucleotides encoding 161P2F10B gene product(s), as well as theisolation of polynucleotides encoding 161P2F10B gene product homologs,alternatively spliced isoforms, allelic variants, and mutant forms of a161P2F10B gene product as well as polynucleotides that encode analogs of161P2F10B-related proteins. Various molecular cloning methods that canbe employed to isolate full length cDNAs encoding a 161P2F10B gene arewell known (see, for example, Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, 2d edition, Cold Spring Harbor Press, New York, 1989;Current Protocols in Molecular Biology. Ausubel et al., Eds., Wiley andSons, 1995). For example, lambda phage cloning methodologies can beconveniently employed, using commercially available cloning systems(e.g., Lambda ZAP Express, Stratagene). Phage clones containing161P2F10B gene cDNAs can be identified by probing with a labeled161P2F10B cDNA or a fragment thereof. For example, in one embodiment, a161P2F10B cDNA (e.g., FIG. 2) or a portion thereof can be synthesizedand used as a probe to retrieve overlapping and full-length cDNAscorresponding to a 161P2F10B gene. A 161P2F10B gene itself can beisolated by screening genomic DNA libraries, bacterial artificialchromosome libraries (BACs), yeast artificial chromosome libraries(YACs), and the like, with 161P2F10B DNA probes or primers.

II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules containinga 161P2F10B polynucleotide, a fragment, analog or homologue thereof,including but not limited to phages, plasmids, phagemids, cosmids, YACs,BACs, as well as various viral and non-viral vectors well known in theart, and cells transformed or transfected with such recombinant DNA orRNA molecules. Methods for generating such molecules are well known(see, for example, Sambrook et al., 1989, supra).

The invention further provides a host-vector system comprising arecombinant DNA molecule containing a 161P2F10B polynucleotide,fragment, analog or homologue thereof within a suitable prokaryotic oreukaryotic host cell. Examples of suitable eukaryotic host cells includea yeast cell, a plant cell, or an animal cell, such as a mammalian cellor an insect cell (e.g., a baculovirus-infectible cell such as an Sf9 orHighFive cell). Examples of suitable mammalian cells include variousprostate cancer cell lines such as DU145 and TsuPr1, other transfectableor transducible prostate cancer cell lines, primary cells (PrEC), aswell as a number of mammalian cells routinely used for the expression ofrecombinant proteins (e.g., COS, CHO, 293, 293T cells). Moreparticularly, a polynucleotide comprising the coding sequence of161P2F10B or a fragment, analog or homolog thereof can be used togenerate 161P2F10B proteins or fragments thereof using any number ofhost-vector systems routinely used and widely known in the art.

A wide range of host-vector systems suitable for the expression of161P2F10B proteins or fragments thereof are available, see for example,Sambrook et al., 1989, supra; Current Protocols in Molecular Biology,1995, supra). Preferred vectors for mammalian expression include but arenot limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviralvector pSRαtkneo (Muller et al., 1991, MCB 11:1785). Using theseexpression vectors, 161P2F10B can be expressed in several prostatecancer and non-prostate cell lines, including for example 293, 293T,rat-1, NIH 3T3 and TsuPr1. The host-vector systems of the invention areuseful for the production of a 161P2F10B protein or fragment thereof.Such host-vector systems can be employed to study the functionalproperties of 161P2F10B and 161P2F10B mutations or analogs.

Recombinant human 161P2F10B protein or an analog or homolog or fragmentthereof can be produced by mammalian cells transfected with a constructencoding a 161P2F10B-related nucleotide. For example, 293T cells can betransfected with an expression plasmid encoding 161P2F10B or fragment,analog or homolog thereof, a 161P2F10B-related protein is expressed inthe 293T cells, and the recombinant 161P2F10B protein is isolated usingstandard purification methods (e.g., affinity purification usinganti-161P2F10B antibodies). In another embodiment, a 161P2F10B codingsequence is subcloned into the retroviral vector pSRαMSVtkneo and usedto infect various mammalian cell lines, such as NIH 3T3, TsuPr1, 293 andrat-1 in order to establish 161P2F10B expressing cell lines. Variousother expression systems well known in the art can also be employed.Expression constructs encoding a leader peptide joined in frame to a161P2F10B coding sequence can be used for the generation of a secretedform of recombinant 161P2F10B protein.

As discussed herein, redundancy in the genetic code permits variation in161P2F10B gene sequences. In particular, it is known in the art thatspecific host species often have specific codon preferences, and thusone can adapt the disclosed sequence as preferred for a desired host.For example, preferred analog codon sequences typically have rare codons(i.e., codons having a usage frequency of less than about 20% in knownsequences of the desired host) replaced with higher frequency codons.Codon preferences for a specific species are calculated, for example, byutilizing codon usage tables available on the INTERNET such as at URLdna.affrc.go.jp/˜nakamura/codon.html.

Additional sequence modifications are known to enhance proteinexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon/intron splice sitesignals, transposon-like repeats, and/or other such well-characterizedsequences that are deleterious to gene expression. The GC content of thesequence is adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Wherepossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures. Other useful modifications include the addition of atranslational initiation consensus sequence at the start of the openreading frame, as described in Kozak, Mol. Cell Biol., 9:5073-5080(1989). Skilled artisans understand that the general rule thateukaryotic ribosomes initiate translation exclusively at the 5′ proximalAUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS92(7): 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)).

III.) 161P2F10B-Related Proteins

Another aspect of the present invention provides 161P2F10B-relatedproteins. Specific embodiments of 161P2F10B proteins comprise apolypeptide having all or part of the amino acid sequence of human161P2F10B as shown in FIG. 2 or FIG. 3. Alternatively, embodiments of161P2F10B proteins comprise variant, homolog or analog polypeptides thathave alterations in the amino acid sequence of 161P2F10B shown in FIG. 2or FIG. 3.

Embodiments of a 161P2F10B polypeptide include: a 161P2F10B polypeptidehaving a sequence shown in FIG. 2, a peptide sequence of a 161P2F10B asshown in FIG. 2 wherein T is U; at least 10 contiguous nucleotides of apolypeptide having the sequence as shown in FIG. 2; or, at least 10contiguous peptides of a polypeptide having the sequence as shown inFIG. 2 where T is U. For example, embodiments of 161P2F10B peptidescomprise, without limitation:

(I) a protein comprising, consisting essentially of, or consisting of anamino acid sequence as shown in FIG. 2A-G or FIG. 3A-E;

(II) a 161P2F10B-related protein that is at least 90, 91, 92, 93, 94,95, 96, 97, 98, 99 or 100% homologous to an entire amino acid sequenceshown in FIG. 2A-G;

(III) a 161P2F10B-related protein that is at least 90, 91, 92, 93, 94,95, 96, 97, 98, 99 or 100% identical to an entire amino acid sequenceshown in FIG. 2A-G or 3A-E;

(IV) a protein that comprises at least one peptide set forth in TablesVIII to XLIX, optionally with a proviso that it is not an entire proteinof FIG. 2;

(V) a protein that comprises at least one peptide set forth in TablesVIII-XXI, collectively, which peptide is also set forth in Tables XXIIto XLIX, collectively, optionally with a proviso that it is not anentire protein of FIG. 2;

(VI) a protein that comprises at least two peptides selected from thepeptides set forth in Tables VIII-XLIX, optionally with a proviso thatit is not an entire protein of FIG. 2;

(VII) a protein that comprises at least two peptides selected from thepeptides set forth in Tables VIII to XLIX collectively, with a provisothat the protein is not a contiguous sequence from an amino acidsequence of FIG. 2;

(VIII) a protein that comprises at least one peptide selected from thepeptides set forth in Tables VIII-XXI; and at least one peptide selectedfrom the peptides set forth in Tables XXII to XLIX, with a proviso thatthe protein is not a contiguous sequence from an amino acid sequence ofFIG. 2;

(IX) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35 amino acids of a protein of FIG. 3A, 3B, 3C, 3D, or 3E inany whole number increment up to 875, 875, 875, 875, or 841 respectivelythat includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35 amino acid position(s) having a value greater than 0.5 in theHydrophilicity profile of FIG. 5;

(X) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35 amino acids of a protein of FIG. 3A, 3B, 3C, 3D, or 3E in anywhole number increment up to 875, 875, 875, 875, or 841 respectively,that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35 amino acid position(s) having a value less than 0.5 in theHydropathicity profile of FIG. 6;

(XI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35 amino acids of a protein of FIG. 3A, 3B, 3C, 3D, or 3E inany whole number increment up to 875, 875, 875, 875, or 841respectively, that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35 amino acid position(s) having a value greaterthan 0.5 in the Percent Accessible Residues profile of FIG. 7;

(XII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35 amino acids of a protein of FIG. 3A, 3B, 3C, 3D, or 3E inany whole number increment up to 875, 875, 875, 875, or 841respectively, that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35 amino acid position(s) having a value greaterthan 0.5 in the Average Flexibility profile of FIG. 8;

(XIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35 amino acids of a protein of FIG. 3A, 3B, 3C, 3D, or 3E inany whole number increment up to 875, 875, 875, 875, or 841respectively, that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35 amino acid position(s) having a value greaterthan 0.5 in the Beta-turn profile of FIG. 9;

(XIV) a peptide that occurs at least twice in Tables VIII-XXI and XXIIto XLIX, collectively;

(XV) a peptide that occurs at least three times in Tables VIII-XXI andXXII to XLIX, collectively;

(XVI) a peptide that occurs at least four times in Tables VIII-XXI andXXII to XLIX, collectively;

(XVII) a peptide that occurs at least five times in Tables VIII-XXI andXXII to XLIX, collectively;

(XVIII) a peptide that occurs at least once in Tables VIII-XXI, and atleast once in tables XXII to XLIX;

(XIX) a peptide that occurs at least once in Tables VIII-XXI, and atleast twice in tables XXII to XLIX;

(XX) a peptide that occurs at least twice in Tables VIII-XXI, and atleast once in tables XXII to XLIX;

(XXI) a peptide that occurs at least twice in Tables VIII-XXI, and atleast twice in tables XXII to XLIX;

(XXII) a peptide which comprises one two, three, four, or five of thefollowing characteristics, or an oligonucleotide encoding such peptide:

-   -   i) a region of at least 5 amino acids of a particular peptide of        FIG. 3, in any whole number increment up to the full length of        that protein in FIG. 3, that includes an amino acid position        having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9,        or having a value equal to 1.0, in the Hydrophilicity profile of        FIG. 5;    -   ii) a region of at least 5 amino acids of a particular peptide        of FIG. 3, in any whole number increment up to the full length        of that protein in FIG. 3, that includes an amino acid position        having a value equal to or less than 0.5, 0.4, 0.3, 0.2, 0.1, or        having a value equal to 0.0, in the Hydropathicity profile of        FIG. 6;    -   iii) a region of at least 5 amino acids of a particular peptide        of FIG. 3, in any whole number increment up to the full length        of that protein in FIG. 3, that includes an amino acid position        having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9,        or having a value equal to 1.0, in the Percent Accessible        Residues profile of FIG. 7;    -   iv) a region of at least 5 amino acids of a particular peptide        of FIG. 3, in any whole number increment up to the full length        of that protein in FIG. 3, that includes an amino acid position        having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9,        or having a value equal to 1.0, in the Average Flexibility        profile of FIG. 8; or,    -   v) a region of at least 5 amino acids of a particular peptide of        FIG. 3, in any whole number increment up to the full length of        that protein in FIG. 3, that includes an amino acid position        having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9,        or having a value equal to 1.0, in the Beta-turn profile of FIG.        9;

(XXIII) a monoloncal antibody or binding region thereof secreted by ahybridoma entitled X41(3)15 deposited with the American Type CultureCollection (ATCC; 10801 University Blvd., Manassas, Va. 20110-2209 USA)on 7 Nov. 2002 and assigned as Patent Deposit Designation No. PTA-4791;

(XXIV) a monoloncal antibody or binding region thereof secreted by ahybridoma entitled X41(3)29 deposited with the American Type CultureCollection (ATCC; 10801 University Blvd., Manassas, Va. 20110-2209 USA)on 7 Nov. 2002 and assigned as Patent Deposit Designation No. PTA-4791;

(XXV) a monoloncal antibody or binding region thereof secreted by ahybridoma entitled X41(3)37 deposited with the American Type CultureCollection (ATCC; 10801 University Blvd., Manassas, Va. 20110-2209 USA)on 7 Nov. 2002 and assigned as Patent Deposit Designation No. PTA-4791;

(XXVI) a monoloncal antibody or binding region thereof secreted by ahybridoma entitled X41(4)6 deposited with the American Type CultureCollection (ATCC; 10801 University Blvd., Manassas, Va. 20110-2209 USA)on 7 Nov. 2002 and assigned as Patent Deposit Designation No. PTA-4794;

(XXVII) a monoloncal antibody or binding region thereof secreted by ahybridoma entitled X41(3)17 deposited with the American Type CultureCollection (ATCC; 10801 University Blvd., Manassas, Va. 20110-2209 USA)on 7 Nov. 2002 and assigned as Patent Deposit Designation No. PTA-4792;

(XXVIII) a monoloncal antibody or binding region thereof secreted by ahybridoma entitled X41(3)50 deposited with the American Type CultureCollection (ATCC; 10801 University Blvd., Manassas, Va. 20110-2209 USA)on 7 Nov. 2002 and assigned as Patent Deposit Designation No. PTA-4793;

(XXIX) a composition comprising a peptide of (I)-(XXII) or an antibodyor binding region thereof of (XXIII to XXVIII) together with apharmaceutical excipient and/or in a human unit dose form.

(XXX) a method of using a peptide of (I)-(XXII), or an antibody orbinding region thereof of (XXIII to XXVIII) or a composition of (XXIX)in a method to modulate a cell expressing 161P2F10b,

(XXXI) a method of using a peptide of (I)-(XXII) or an antibody orbinding region thereof of (XXIII to XXVIII) or a composition of (XXIX)in a method to diagnose, prophylax, prognose, or treat an individual whobears a cell expressing 161P2F10b

(XXXII) a method of using a peptide of (I)-(XXII) or an antibody orbinding region thereof of (XXIII to XXVIII) or a composition (XXIX) in amethod to diagnose, prophylax, prognose, or treat an individual whobears a cell expressing 161P2F10b, said cell from a cancer of a tissuelisted in Table I;

(XXXIII) a method of using a peptide of (I)-(XXII) or an antibody orbinding region thereof of (XXIII to XXVIII) or a composition of (XXIX)in a method to diagnose, prophylax, prognose, or treat a cancer;

(XXXIV) a method of using a peptide of (I)-(XXII) or an antibody orbinding region thereof of (XXIII to XXVIII) or a composition of (XXIX)in a method to diagnose, prophylax, prognose, or treat a cancer of atissue listed in Table I; and,

(XXXV) a method of using a peptide of (I)-(XXII) or an antibody orbinding region thereof of (XXIII to XXVIII) or a composition (XXIX) in amethod to identify or characterize a modulator of a cell expressing161P2F10b.

As used herein, a range is understood to specifically disclose all wholeunit positions thereof.

Typical embodiments of the invention disclosed herein include 161P2F10Bpolynucleotides that encode specific portions of 161P2F10B mRNAsequences (and those which are complementary to such sequences) such asthose that encode the proteins and/or fragments thereof, for example:

(a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165,170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375,400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725,750, 775, 800, 825, 850, 860, 870, 875 or more contiguous amino acids of161P2F10B variant 1; the maximal lengths relevant for other variantsare: variant 2, 875 amino acids; variant 3, 875 amino acids, variant 4,875, and variant 7, 841 amino acids.

In general, naturally occurring allelic variants of human 161P2F10Bshare a high degree of structural identity and homology (e.g., 90% ormore homology). Typically, allelic variants of a 161P2F10B proteincontain conservative amino acid substitutions within the 161P2F10Bsequences described herein or contain a substitution of an amino acidfrom a corresponding position in a homologue of 161P2F10B. One class of161P2F10B allelic variants are proteins that share a high degree ofhomology with at least a small region of a particular 161P2F10B aminoacid sequence, but further contain a radical departure from thesequence, such as a non-conservative substitution, truncation, insertionor frame shift. In comparisons of protein sequences, the terms,similarity, identity, and homology each have a distinct meaning asappreciated in the field of genetics. Moreover, orthology and paralogycan be important concepts describing the relationship of members of agiven protein family in one organism to the members of the same familyin other organisms.

Amino acid abbreviations are provided in Table II. Conservative aminoacid substitutions can frequently be made in a protein without alteringeither the conformation or the function of the protein. Proteins of theinvention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15conservative substitutions. Such changes include substituting any ofisoleucine (I), valine (V), and leucine (L) for any other of thesehydrophobic amino acids; aspartic acid (D) for glutamic acid (E) andvice versa; glutamine (Q) for asparagine (N) and vice versa; and serine(S) for threonine (T) and vice versa. Other substitutions can also beconsidered conservative, depending on the environment of the particularamino acid and its role in the three-dimensional structure of theprotein. For example, glycine (G) and alanine (A) can frequently beinterchangeable, as can alanine (A) and valine (V). Methionine (M),which is relatively hydrophobic, can frequently be interchanged withleucine and isoleucine, and sometimes with valine. Lysine (K) andarginine (R) are frequently interchangeable in locations in which thesignificant feature of the amino acid residue is its charge and thediffering pK's of these two amino acid residues are not significant.Still other changes can be considered “conservative” in particularenvironments (see, e.g. Table III herein; pages 13-15 “Biochemistry”2^(nd) ED. Lubert Stryer ed (Stanford University); Henikoff et al., PNAS1992 Vol 89 10915-10919; Lei et al., J Biol Chem 1995 May 19;270(20):11882-6).

Embodiments of the invention disclosed herein include a wide variety ofart-accepted variants or analogs of 161P2F10B proteins such aspolypeptides having amino acid insertions, deletions and substitutions.161P2F10B variants can be made using methods known in the art such assite-directed mutagenesis, alanine scanning, and PCR mutagenesis.Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331(1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassettemutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selectionmutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415(1986)) or other known techniques can be performed on the cloned DNA toproduce the 161P2F10B variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence that is involved in aspecific biological activity such as a protein-protein interaction.Among the preferred scanning amino acids are relatively small, neutralamino acids. Such amino acids include alanine, glycine, serine, andcysteine. Alanine is typically a preferred scanning amino acid amongthis group because it eliminates the side-chain beyond the beta-carbonand is less likely to alter the main-chain conformation of the variant.Alanine is also typically preferred because it is the most common aminoacid. Further, it is frequently found in both buried and exposedpositions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia,J. Mol. Biol., 150:1 (1976)). If alanine substitution does not yieldadequate amounts of variant, an isosteric amino acid can be used.

As defined herein, 161P2F10B variants, analogs or homologs, have thedistinguishing attribute of having at least one epitope that is “crossreactive” with a 161P2F10B protein having an amino acid sequence of FIG.3. As used in this sentence, “cross reactive” means that an antibody orT cell that specifically binds to a 161P2F10B variant also specificallybinds to a 161P2F10B protein having an amino acid sequence set forth inFIG. 3. A polypeptide ceases to be a variant of a protein shown in FIG.3, when it no longer contains any epitope capable of being recognized byan antibody or T cell that specifically binds to the starting 161P2F10Bprotein. Those skilled in the art understand that antibodies thatrecognize proteins bind to epitopes of varying size, and a grouping ofthe order of about four or five amino acids, contiguous or not, isregarded as a typical number of amino acids in a minimal epitope. See,e.g., Nair et al., J. Immunol 2000 165(12): 6949-6955; Hebbes et al.,Mol Immunol (1989) 26(9):865-73; Schwartz et al., J Immunol (1985)135(4):2598-608.

Other classes of 161P2F10B-related protein variants share 70%, 75%, 80%,85% or 90% or more similarity with an amino acid sequence of FIG. 3, ora fragment thereof. Another specific class of 161P2F10B protein variantsor analogs comprises one or more of the 161P2F10B biological motifsdescribed herein or presently known in the art. Thus, encompassed by thepresent invention are analogs of 161P2F10B fragments (nucleic or aminoacid) that have altered functional (e g immunogenic) properties relativeto the starting fragment. It is to be appreciated that motifs now orwhich become part of the art are to be applied to the nucleic or aminoacid sequences of FIG. 2 or FIG. 3.

As discussed herein, embodiments of the claimed invention includepolypeptides containing less than the full amino acid sequence of a161P2F10B protein shown in FIG. 2 or FIG. 3. For example, representativeembodiments of the invention comprise peptides/proteins having any 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of a161P2F10B protein shown in FIG. 2 or FIG. 3.

Moreover, representative embodiments of the invention disclosed hereininclude polypeptides consisting of about amino acid 1 to about aminoacid 10 of a 161P2F10B protein shown in FIG. 2 or FIG. 3, polypeptidesconsisting of about amino acid 10 to about amino acid 20 of a 161P2F10Bprotein shown in FIG. 2 or FIG. 3, polypeptides consisting of aboutamino acid 20 to about amino acid 30 of a 161P2F10B protein shown inFIG. 2 or FIG. 3, polypeptides consisting of about amino acid 30 toabout amino acid 40 of a 161P2F10B protein shown in FIG. 2 or FIG. 3,polypeptides consisting of about amino acid 40 to about amino acid 50 ofa 161P2F10B protein shown in FIG. 2 or FIG. 3, polypeptides consistingof about amino acid 50 to about amino acid 60 of a 161P2F10B proteinshown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid60 to about amino acid 70 of a 161P2F10B protein shown in FIG. 2 or FIG.3, polypeptides consisting of about amino acid 70 to about amino acid 80of a 161P2F10B protein shown in FIG. 2 or FIG. 3, polypeptidesconsisting of about amino acid 80 to about amino acid 90 of a 161P2F10Bprotein shown in FIG. 2 or FIG. 3, polypeptides consisting of aboutamino acid 90 to about amino acid 100 of a 161P2F10B protein shown inFIG. 2 or FIG. 3, etc. throughout the entirety of a 161P2F10B amino acidsequence. Moreover, polypeptides consisting of about amino acid 1 (or 20or 30 or 40 etc.) to about amino acid 20, (or 130, or 140 or 150 etc.)of a 161P2F10B protein shown in FIG. 2 or FIG. 3 are embodiments of theinvention. It is to be appreciated that the starting and stoppingpositions in this paragraph refer to the specified position as well asthat position plus or minus 5 residues.

161P2F10B-related proteins are generated using standard peptidesynthesis technology or using chemical cleavage methods well known inthe art. Alternatively, recombinant methods can be used to generatenucleic acid molecules that encode a 161P2F10B-related protein. In oneembodiment, nucleic acid molecules provide a means to generate definedfragments of a 161P2F10B protein (or variants, homologs or analogsthereof).

III.A.) Motif-Bearing Protein Embodiments

Additional illustrative embodiments of the invention disclosed hereininclude 161P2F10B polypeptides comprising the amino acid residues of oneor more of the biological motifs contained within a 161P2F10Bpolypeptide sequence set forth in FIG. 2 or FIG. 3. Various motifs areknown in the art, and a protein can be evaluated for the presence ofsuch motifs by a number of publicly available Internet sites (see, e.g.,Epimatrix™ and Epimer).

Motif bearing subsequences of all 161P2F10B variant proteins are setforth and identified in Tables VIII-XXI and XXII-XLIX.

Table V sets forth several frequently occurring motifs based on pfamsearches (see URL address pfam.wustl.edu/). The columns of Table V list(1) motif name abbreviation, (2) percent identity found amongst thedifferent member of the motif family, (3) motif name or description and(4) most common function; location information is included if the motifis relevant for location.

Polypeptides comprising one or more of the 161P2F10B motifs discussedabove are useful in elucidating the specific characteristics of amalignant phenotype in view of the observation that the 161P2F10B motifsdiscussed above are associated with growth dysregulation and because161P2F10B is overexpressed in certain cancers (See, e.g., Table I).Casein kinase II, cAMP and camp-dependent protein kinase, and ProteinKinase C, for example, are enzymes known to be associated with thedevelopment of the malignant phenotype (see e.g. Chen et al., LabInvest., 78(2): 165-174 (1998); Gaiddon et al., Endocrinology 136(10):4331-4338 (1995); Hall et al., Nucleic Acids Research 24(6): 1119-1126(1996); Peterziel et al., Oncogene 18(46): 6322-6329 (1999) and O'Brian,Oncol. Rep. 5(2): 305-309 (1998)). Moreover, both glycosylation andmyristoylation are protein modifications also associated with cancer andcancer progression (see e.g. Dennis et al., Biochem. Biophys. Acta1473(1):21-34 (1999); Raju et al., Exp. Cell Res. 235(1): 145-154(1997)). Amidation is another protein modification also associated withcancer and cancer progression (see e.g. Treston et al., J. Natl. CancerInst. Monogr. (13): 169-175 (1992)).

In another embodiment, proteins of the invention comprise one or more ofthe immunoreactive epitopes identified in accordance with art-acceptedmethods, such as the peptides set forth in Tables VIII-XXI andXXII-XLIX. CTL epitopes can be determined using specific algorithms toidentify peptides within a 161P2F10B protein that are capable ofoptimally binding to specified HLA alleles (e.g., Table IV; Epimatrix™and Epimer™, and BIMAS). Moreover, processes for identifying peptidesthat have sufficient binding affinity for HLA molecules and which arecorrelated with being immunogenic epitopes, are well known in the art,and are carried out without undue experimentation. In addition,processes for identifying peptides that are immunogenic epitopes, arewell known in the art, and are carried out without undue experimentationeither in vitro or in vivo.

Also known in the art are principles for creating analogs of suchepitopes in order to modulate immunogenicity. For example, one beginswith an epitope that bears a CTL or HTL motif (see, e.g., the HLA ClassI and HLA Class II motifs/supermotifs of Table IV). The epitope isanaloged by substituting out an amino acid at one of the specifiedpositions, and replacing it with another amino acid specified for thatposition. For example, on the basis of residues defined in Table IV, onecan substitute out a deleterious residue in favor of any other residue,such as a preferred residue; substitute a less-preferred residue with apreferred residue; or substitute an originally-occurring preferredresidue with another preferred residue. Substitutions can occur atprimary anchor positions or at other positions in a peptide; see, e.g.,Table IV.

A variety of references reflect the art regarding the identification andgeneration of epitopes in a protein of interest as well as analogsthereof. See, for example, WO 97/33602 to Chesnut et al.; Sette,Immunogenetics 1999 50(3-4): 201-212; Sette et al., J. Immunol. 2001166(2): 1389-1397; Sidney et al., Hum. Immunol. 1997 58(1): 12-20; Kondoet al., Immunogenetics 1997 45(4): 249-258; Sidney et al., J. Immunol.1996 157(8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt etal., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7(1992); Parker et al., J. Immunol. 152:163-75 (1994)); Kast et al., 1994152(8): 3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3):266-278; Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633;Alexander et al., PMID: 7895164, UI: 95202582; O′Sullivan et al., J.Immunol. 1991 147(8): 2663-2669; Alexander et al., Immunity 1994 1(9):751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92.

Related embodiments of the invention include polypeptides comprisingcombinations of the different motifs set forth in Table VI, and/or, oneor more of the predicted CTL epitopes of Tables VIII-XXI and XXII-XLIX,and/or, one or more of the predicted HTL epitopes of Tables XLVI-XLIX,and/or, one or more of the T cell binding motifs known in the art.Preferred embodiments contain no insertions, deletions or substitutionseither within the motifs or within the intervening sequences of thepolypeptides. In addition, embodiments which include a number of eitherN-terminal and/or C-terminal amino acid residues on either side of thesemotifs may be desirable (to, for example, include a greater portion ofthe polypeptide architecture in which the motif is located). Typically,the number of N-terminal and/or C-terminal amino acid residues on eitherside of a motif is between about 1 to about 100 amino acid residues,preferably 5 to about 50 amino acid residues.

161P2F10B-related proteins are embodied in many forms, preferably inisolated form. A purified 161P2F10B protein molecule will besubstantially free of other proteins or molecules that impair thebinding of 161P2F10B to antibody, T cell or other ligand. The nature anddegree of isolation and purification will depend on the intended use.Embodiments of a 161P2F10B-related proteins include purified161P2F10B-related proteins and functional, soluble 161P2F10B-relatedproteins. In one embodiment, a functional, soluble 161P2F10B protein orfragment thereof retains the ability to be bound by antibody, T cell orother ligand.

The invention also provides 161P2F10B proteins comprising biologicallyactive fragments of a 161P2F10B amino acid sequence shown in FIG. 2 orFIG. 3. Such proteins exhibit properties of the starting 161P2F10Bprotein, such as the ability to elicit the generation of antibodies thatspecifically bind an epitope associated with the starting 161P2F10Bprotein; to be bound by such antibodies; to elicit the activation of HTLor CTL; and/or, to be recognized by HTL or CTL that also specificallybind to the starting protein.

161P2F10B-related polypeptides that contain particularly interestingstructures can be predicted and/or identified using various analyticaltechniques well known in the art, including, for example, the methods ofChou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultzor Jameson-Wolf analysis, or based on immunogenicity. Fragments thatcontain such structures are particularly useful in generatingsubunit-specific anti-161P2F10B antibodies or T cells or in identifyingcellular factors that bind to 161P2F10B. For example, hydrophilicityprofiles can be generated, and immunogenic peptide fragments identified,using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl.Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can begenerated, and immunogenic peptide fragments identified, using themethod of Kyte, J. and Doolittle, R. F., 1982, J. Mol. Biol.157:105-132. Percent (%) Accessible Residues profiles can be generated,and immunogenic peptide fragments identified, using the method of JaninJ., 1979, Nature 277:491-492. Average Flexibility profiles can begenerated, and immunogenic peptide fragments identified, using themethod of Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. ProteinRes. 32:242-255. Beta-turn profiles can be generated, and immunogenicpeptide fragments identified, using the method of Deleage, G., Roux B.,1987, Protein Engineering 1:289-294.

CTL epitopes can be determined using specific algorithms to identifypeptides within a 161P2F10B protein that are capable of optimallybinding to specified HLA alleles (e.g., by using the SYFPEITHI site atWorld Wide Web; the listings in Table IV(A)-(E); Epimatrix™ and Epimer™,and BIMAS). Illustrating this, peptide epitopes from 161P2F10B that arepresented in the context of human MHC Class I molecules, e.g., HLA-A1,A2, A3, A11, A24, B7 and B35 were predicted (see, e.g., Tables VIII-XXI,XXII-XLIX). Specifically, the complete amino acid sequence of the161P2F10B protein and relevant portions of other variants, i.e., for HLAClass I predictions 9 flanking residues on either side of a pointmutation or exon junction, and for HLA Class II predictions 14 flankingresidues on either side of a point mutation or exon junctioncorresponding to that variant, were entered into the HLA Peptide MotifSearch algorithm found in the Bioinformatics and Molecular AnalysisSection (BIMAS) web site listed above; in addition to the siteSYFPEITHI.

The HLA peptide motif search algorithm was developed by Dr. Ken Parkerbased on binding of specific peptide sequences in the groove of HLAClass I molecules, in particular HLA-A2 (see, e.g., Falk et al., Nature351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker etal., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol.152:163-75 (1994)). This algorithm allows location and ranking of 8-mer,9-mer, and 10-mer peptides from a complete protein sequence forpredicted binding to HLA-A2 as well as numerous other HLA Class Imolecules. Many HLA class I binding peptides are 8-, 9-, 10 or 11-mers.For example, for Class I HLA-A2, the epitopes preferably contain aleucine (L) or methionine (M) at position 2 and a valine (V) or leucine(L) at the C-terminus (see, e.g., Parker et al., J. Immunol. 149:3580-7(1992)). Selected results of 161P2F10B predicted binding peptides areshown in Tables VIII-XXI and XXII-XLIX herein. In Tables VIII-XXI andXXII-XLVII, selected candidates, 9-mers and 10-mers, for each familymember are shown along with their location, the amino acid sequence ofeach specific peptide, and an estimated binding score. In TablesXLVI-XLIX, selected candidates, 15-mers, for each family member areshown along with their location, the amino acid sequence of eachspecific peptide, and an estimated binding score. The binding scorecorresponds to the estimated half time of dissociation of complexescontaining the peptide at 37° C. at pH 6.5. Peptides with the highestbinding score are predicted to be the most tightly bound to HLA Class Ion the cell surface for the greatest period of time and thus representthe best immunogenic targets for T-cell recognition.

Actual binding of peptides to an HLA allele can be evaluated bystabilization of HLA expression on the antigen-processing defective cellline T2 (see, e.g., Xue et al., Prostate 30:73-8 (1997) and Peshwa etal., Prostate 36:129-38 (1998)) Immunogenicity of specific peptides canbe evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes(CTL) in the presence of antigen presenting cells such as dendriticcells.

It is to be appreciated that every epitope predicted by the BIMAS site,Epimer™ and Epimatrix™ sites, or specified by the HLA class I or classII motifs available in the art or which become part of the art such asset forth in Table IV (see, e.g., SYFPEITHI or BIMAS web sites) are tobe “applied” to a 161P2F10B protein in accordance with the invention. Asused in this context “applied” means that a 161P2F10B protein isevaluated, e.g., visually or by computer-based patterns finding methods,as appreciated by those of skill in the relevant art. Every subsequenceof a 161P2F10B protein of 8, 9, 10, or 11 amino acid residues that bearsan HLA Class I motif, or a subsequence of 9 or more amino acid residuesthat bear an HLA Class II motif are within the scope of the invention.

III.B.) Expression of 161P2F10B-Related Proteins

In an embodiment described in the examples that follow, 161P2F10B can beconveniently expressed in cells (such as 293T cells) transfected with acommercially available expression vector such as a CMV-driven expressionvector encoding 161P2F10B with a C-terminal 6×His and MYC tag(pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, NashvilleTenn.). The Tag5 vector provides an IgGK secretion signal that can beused to facilitate the production of a secreted 161P2F10B protein intransfected cells. The secreted HIS-tagged 161P2F10B in the culturemedia can be purified, e.g., using a nickel column using standardtechniques.

III.C.) Modifications of 161P2F10B-Related Proteins

Modifications of 161P2F10B-related proteins such as covalentmodifications are included within the scope of this invention. One typeof covalent modification includes reacting targeted amino acid residuesof a 161P2F10B polypeptide with an organic derivatizing agent that iscapable of reacting with selected side chains or the N- or C-terminalresidues of a 161P2F10B protein. Another type of covalent modificationof a 161P2F10B polypeptide included within the scope of this inventioncomprises altering the native glycosylation pattern of a protein of theinvention. Another type of covalent modification of 161P2F10B compriseslinking a 161P2F10B polypeptide to one of a variety of nonproteinaceouspolymers, e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The 161P2F10B-related proteins of the present invention can also bemodified to form a chimeric molecule comprising 161P2F10B fused toanother, heterologous polypeptide or amino acid sequence. Such achimeric molecule can be synthesized chemically or recombinantly. Achimeric molecule can have a protein of the invention fused to anothertumor-associated antigen or fragment thereof. Alternatively, a proteinin accordance with the invention can comprise a fusion of fragments of a161P2F10B sequence (amino or nucleic acid) such that a molecule iscreated that is not, through its length, directly homologous to theamino or nucleic acid sequences shown in FIG. 2 or FIG. 3. Such achimeric molecule can comprise multiples of the same subsequence of161P2F10B. A chimeric molecule can comprise a fusion of a161P2F10B-related protein with a polyhistidine epitope tag, whichprovides an epitope to which immobilized nickel can selectively bind,with cytokines or with growth factors. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of a 161P2F10B protein. In analternative embodiment, the chimeric molecule can comprise a fusion of a161P2F10B-related protein with an immunoglobulin or a particular regionof an immunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a 161P2F10B polypeptide in place of at least one variable regionwithin an Ig molecule. In a preferred embodiment, the immunoglobulinfusion includes the hinge, CH2 and CH3, or the hinge, CHI, CH2 and CH3regions of an IgGI molecule. For the production of immunoglobulinfusions see, e.g., U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.

III.D.) Uses of 161P2F10B-Related Proteins

The proteins of the invention have a number of different specific uses.As 161P2F10B is highly expressed in prostate and other cancers,161P2F10B-related proteins are used in methods that assess the status of161P2F10B gene products in normal versus cancerous tissues, therebyelucidating the malignant phenotype. Typically, polypeptides fromspecific regions of a 161P2F10B protein are used to assess the presenceof perturbations (such as deletions, insertions, point mutations etc.)in those regions (such as regions containing one or more motifs).Exemplary assays utilize antibodies or T cells targeting161P2F10B-related proteins comprising the amino acid residues of one ormore of the biological motifs contained within a 161P2F10B polypeptidesequence in order to evaluate the characteristics of this region innormal versus cancerous tissues or to elicit an immune response to theepitope. Alternatively, 161P2F10B-related proteins that contain theamino acid residues of one or more of the biological motifs in a161P2F10B protein are used to screen for factors that interact with thatregion of 161P2F10B.

161P2F10B protein fragments/subsequences are particularly useful ingenerating and characterizing domain-specific antibodies (e.g.,antibodies recognizing an extracellular or intracellular epitope of a161P2F10B protein), for identifying agents or cellular factors that bindto 161P2F10B or a particular structural domain thereof, and in varioustherapeutic and diagnostic contexts, including but not limited todiagnostic assays, cancer vaccines and methods of preparing suchvaccines.

Proteins encoded by the 161P2F10B genes, or by analogs, homologs orfragments thereof, have a variety of uses, including but not limited togenerating antibodies and in methods for identifying ligands and otheragents and cellular constituents that bind to a 161P2F10B gene product.Antibodies raised against a 161P2F10B protein or fragment thereof areuseful in diagnostic and prognostic assays, and imaging methodologies inthe management of human cancers characterized by expression of 161P2F10Bprotein, such as those listed in Table I. Such antibodies can beexpressed intracellularly and used in methods of treating patients withsuch cancers. 161P2F10B-related nucleic acids or proteins are also usedin generating HTL or CTL responses.

Various immunological assays useful for the detection of 161P2F10Bproteins are used, including but not limited to various types ofradioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), immunocytochemicalmethods, and the like. Antibodies can be labeled and used asimmunological imaging reagents capable of detecting 161P2F10B-expressingcells (e.g., in radioscintigraphic imaging methods). 161P2F10B proteinsare also particularly useful in generating cancer vaccines, as furtherdescribed herein.

IV.) 161P2F10B Antibodies

Another aspect of the invention provides antibodies that bind to161P2F10B-related proteins. Preferred antibodies specifically bind to a161P2F10B-related protein and do not bind (or bind weakly) to peptidesor proteins that are not 161P2F10B-related proteins. For example,antibodies that bind 161P2F10B can bind 161P2F10B-related proteins suchas the homologs or analogs thereof.

161P2F10B antibodies of the invention are particularly useful in cancer(see, e.g., Table I) diagnostic and prognostic assays, and imagingmethodologies. Similarly, such antibodies are useful in the treatment,diagnosis, and/or prognosis of other cancers, to the extent 161P2F10B isalso expressed or overexpressed in these other cancers. Moreover,intracellularly expressed antibodies (e.g., single chain antibodies) aretherapeutically useful in treating cancers in which the expression of161P2F10B is involved, such as advanced or metastatic prostate cancers.

The invention also provides various immunological assays useful for thedetection and quantification of 161P2F10B and mutant 161P2F10B-relatedproteins. Such assays can comprise one or more 161P2F10B antibodiescapable of recognizing and binding a 161P2F10B-related protein, asappropriate. These assays are performed within various immunologicalassay formats well known in the art, including but not limited tovarious types of radioimmunoassays, enzyme-linked immunosorbent assays(ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.

Immunological non-antibody assays of the invention also comprise T cellimmunogenicity assays (inhibitory or stimulatory) as well as majorhistocompatibility complex (MHC) binding assays.

In addition, immunological imaging methods capable of detecting prostatecancer and other cancers expressing 161P2F10B are also provided by theinvention, including but not limited to radioscintigraphic imagingmethods using labeled 161P2F10B antibodies. Such assays are clinicallyuseful in the detection, monitoring, and prognosis of 161P2F10Bexpressing cancers such as prostate cancer.

161P2F10B antibodies are also used in methods for purifying a161P2F10B-related protein and for isolating 161P2F10B homologues andrelated molecules. For example, a method of purifying a161P2F10B-related protein comprises incubating a 161P2F10B antibody,which has been coupled to a solid matrix, with a lysate or othersolution containing a 161P2F10B-related protein under conditions thatpermit the 161P2F10B antibody to bind to the 161P2F10B-related protein;washing the solid matrix to eliminate impurities; and eluting the161P2F10B-related protein from the coupled antibody. Other uses of161P2F10B antibodies in accordance with the invention include generatinganti-idiotypic antibodies that mimic a 161P2F10B protein.

Various methods for the preparation of antibodies are well known in theart. For example, antibodies can be prepared by immunizing a suitablemammalian host using a 161P2F10B-related protein, peptide, or fragment,in isolated or immunoconjugated form (Antibodies: A Laboratory Manual,CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, ColdSpring Harbor Press, NY (1989)). In addition, fusion proteins of161P2F10B can also be used, such as a 161P2F10B GST-fusion protein. In aparticular embodiment, a GST fusion protein comprising all or most ofthe amino acid sequence of FIG. 2 or FIG. 3 is produced, then used as animmunogen to generate appropriate antibodies. In another embodiment, a161P2F10B-related protein is synthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art are used(with or without purified 161P2F10B-related protein or 161P2F10Bexpressing cells) to generate an immune response to the encodedimmunogen (for review, see Donnelly et al., 1997, Ann Rev. Immunol. 15:617-648).

The amino acid sequence of a 161P2F10B protein as shown in FIG. 2 orFIG. 3 can be analyzed to select specific regions of the 161P2F10Bprotein for generating antibodies. For example, hydrophobicity andhydrophilicity analyses of a 161P2F10B amino acid sequence are used toidentify hydrophilic regions in the 161P2F10B structure. Regions of a161P2F10B protein that show immunogenic structure, as well as otherregions and domains, can readily be identified using various othermethods known in the art, such as Chou-Fasman, Garnier-Robson,Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis.Hydrophilicity profiles can be generated using the method of Hopp, T. P.and Woods, K. R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828.Hydropathicity profiles can be generated using the method of Kyte, J.and Doolittle, R. F., 1982, J. Mol. Biol. 157:105-132. Percent (%)Accessible Residues profiles can be generated using the method of JaninJ., 1979, Nature 277:491-492. Average Flexibility profiles can begenerated using the method of Bhaskaran R., Ponnuswamy P. K., 1988, Int.J. Pept. Protein Res. 32:242-255. Beta-turn profiles can be generatedusing the method of Deleage, G., Roux B., 1987, Protein Engineering1:289-294. Thus, each region identified by any of these programs ormethods is within the scope of the present invention. Methods for thegeneration of 161P2F10B antibodies are further illustrated by way of theexamples provided herein. Methods for preparing a protein or polypeptidefor use as an immunogen are well known in the art. Also well known inthe art are methods for preparing immunogenic conjugates of a proteinwith a carrier, such as BSA, KLH or other carrier protein. In somecircumstances, direct conjugation using, for example, carbodiimidereagents are used; in other instances linking reagents such as thosesupplied by Pierce Chemical Co., Rockford, Ill., are effective.Administration of a 161P2F10B immunogen is often conducted by injectionover a suitable time period and with use of a suitable adjuvant, as isunderstood in the art. During the immunization schedule, titers ofantibodies can be taken to determine adequacy of antibody formation.

161P2F10B monoclonal antibodies can be produced by various means wellknown in the art. For example, immortalized cell lines that secrete adesired monoclonal antibody are prepared using the standard hybridomatechnology of Kohler and Milstein or modifications that immortalizeantibody-producing B cells, as is generally known Immortalized celllines that secrete the desired antibodies are screened by immunoassay inwhich the antigen is a 161P2F10B-related protein. When the appropriateimmortalized cell culture is identified, the cells can be expanded andantibodies produced either from in vitro cultures or from ascites fluid.

The antibodies or fragments of the invention can also be produced, byrecombinant means. Regions that bind specifically to the desired regionsof a 161P2F10B protein can also be produced in the context of chimericor complementarity-determining region (CDR) grafted antibodies ofmultiple species origin. Humanized or human 161P2F10B antibodies canalso be produced, and are preferred for use in therapeutic contexts.Methods for humanizing murine and other non-human antibodies, bysubstituting one or more of the non-human antibody CDRs forcorresponding human antibody sequences, are well known (see for example,Jones et al., 1986, Nature 321: 522-525; Riechmann et al., 1988, Nature332: 323-327; Verhoeyen et al., 1988, Science 239: 1534-1536). See also,Carter et al., 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and Sims etal., 1993, J. Immunol. 151: 2296.

Methods for producing fully human monoclonal antibodies include phagedisplay and transgenic methods (for review, see Vaughan et al., 1998,Nature Biotechnology 16: 535-539). Fully human 161P2F10B monoclonalantibodies can be generated using cloning technologies employing largehuman Ig gene combinatorial libraries (i.e., phage display) (Griffithsand Hoogenboom, Building an in vitro immune system: human antibodiesfrom phage display libraries. In: Protein Engineering of AntibodyMolecules for Prophylactic and Therapeutic Applications in Man, Clark,M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, HumanAntibodies from combinatorial libraries. Id., pp 65-82). Fully human161P2F10B monoclonal antibodies can also be produced using transgenicmice engineered to contain human immunoglobulin gene loci as describedin PCT Patent Application WO98/24893, Kucherlapati and Jakobovits etal., published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin.Invest. Drugs 7(4): 607-614; U.S. Pat. No. 6,162,963 issued 19 Dec.2000; U.S. Pat. No. 6,150,584 issued 12 Nov. 2000; and, U.S. Pat. No.6,114,598 issued 5 Sep. 2000). This method avoids the in vitromanipulation required with phage display technology and efficientlyproduces high affinity authentic human antibodies.

Reactivity of 161P2F10B antibodies with a 161P2F10B-related protein canbe established by a number of well known means, including Western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,161P2F10B-related proteins, 161P2F10B-expressing cells or extractsthereof. A 161P2F10B antibody or fragment thereof can be labeled with adetectable marker or conjugated to a second molecule. Suitabledetectable markers include, but are not limited to, a radioisotope, afluorescent compound, a bioluminescent compound, chemiluminescentcompound, a metal chelator or an enzyme. Further, bi-specific antibodiesspecific for two or more 161P2F10B epitopes are generated using methodsgenerally known in the art. Homodimeric antibodies can also be generatedby cross-linking techniques known in the art (e.g., Wolff et al., CancerRes. 53: 2560-2565).

V.) 161P2F10B Cellular Immune Responses

The mechanism by which T cells recognize antigens has been delineated.Efficacious peptide epitope vaccine compositions of the invention inducea therapeutic or prophylactic immune responses in very broad segments ofthe world-wide population. For an understanding of the value andefficacy of compositions of the invention that induce cellular immuneresponses, a brief review of immunology-related technology is provided.

A complex of an HLA molecule and a peptidic antigen acts as the ligandrecognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071,1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A. andBodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev.Immunol. 11:403, 1993). Through the study of single amino acidsubstituted antigen analogs and the sequencing of endogenously bound,naturally processed peptides, critical residues that correspond tomotifs required for specific binding to HLA antigen molecules have beenidentified and are set forth in Table IV (see also, e.g., Southwood, etal., J. Immunol. 160:3363, 1998; Rammensee, et al., Immunogenetics41:178, 1995; Rammensee et al., SYFPEITHI, access via World Wide Web;Sette, A. and Sidney, J. Curr. Opin. Immunol. 10:478, 1998; Engelhard,V. H., Curr. Opin. Immunol. 6:13, 1994; Sette, A. and Grey, H. M., Curr.Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J. Curr. Biol.6:52, 1994; Ruppert et al., Cell 74:929-937, 1993; Kondo et al., J.Immunol. 155:4307-4312, 1995; Sidney et al., J. Immunol. 157:3480-3490,1996; Sidney et al., Human Immunol. 45:79-93, 1996; Sette, A. andSidney, J. Immunogenetics 1999 November; 50(3-4):201-12, Review).

Furthermore, x-ray crystallographic analyses of HLA-peptide complexeshave revealed pockets within the peptide binding cleft/groove of HLAmolecules which accommodate, in an allele-specific mode, residues borneby peptide ligands; these residues in turn determine the HLA bindingcapacity of the peptides in which they are present. (See, e.g., Madden,D. R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity 4:203,1996; Fremont et al., Immunity 8:305, 1998; Stern et al., Structure2:245, 1994; Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H.et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci.USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M.L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927,1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science257:919, 1992; Saper, M. A. , Bjorkman, P. J. and Wiley, D. C., J. Mol.Biol. 219:277, 1991.)

Accordingly, the definition of class I and class II allele-specific HLAbinding motifs, or class I or class II supermotifs allows identificationof regions within a protein that are correlated with binding toparticular HLA antigen(s).

Thus, by a process of HLA motif identification, candidates forepitope-based vaccines have been identified; such candidates can befurther evaluated by HLA-peptide binding assays to determine bindingaffinity and/or the time period of association of the epitope and itscorresponding HLA molecule. Additional confirmatory work can beperformed to select, amongst these vaccine candidates, epitopes withpreferred characteristics in terms of population coverage, and/orimmunogenicity.

Various strategies can be utilized to evaluate cellular immunogenicity,including:

1) Evaluation of primary T cell cultures from normal individuals (see,e.g., Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995; Celis, E. etal., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J.Immunol. 158:1796, 1997; Kawashima, I. et al., Human Immunol. 59:1,1998). This procedure involves the stimulation of peripheral bloodlymphocytes (PBL) from normal subjects with a test peptide in thepresence of antigen presenting cells in vitro over a period of severalweeks. T cells specific for the peptide become activated during thistime and are detected using, e.g., a lymphokine- or ⁵¹ Cr-release assayinvolving peptide sensitized target cells.

2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. etal., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol.8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997). Forexample, in such methods peptides in incomplete Freund's adjuvant areadministered subcutaneously to HLA transgenic mice. Several weeksfollowing immunization, splenocytes are removed and cultured in vitro inthe presence of test peptide for approximately one week.Peptide-specific T cells are detected using, e.g., a ⁵¹ Cr-release assayinvolving peptide sensitized target cells and target cells expressingendogenously generated antigen.

3) Demonstration of recall T cell responses from immune individuals whohave been either effectively vaccinated and/or from chronically illpatients (see, e.g., Rehermann, B. et al., J. Exp. Med. 181:1047, 1995;Doolan, D. L. et al., Immunity 7:97, 1997; Bertoni, R. et al., J. Clin.Invest. 100:503, 1997; Threlkeld, S. C. et al., J. Immunol. 159:1648,1997; Diepolder, H. M. et al., J. Virol. 71:6011, 1997). Accordingly,recall responses are detected by culturing PBL from subjects that havebeen exposed to the antigen due to disease and thus have generated animmune response “naturally”, or from patients who were vaccinatedagainst the antigen. PBL from subjects are cultured in vitro for 1-2weeks in the presence of test peptide plus antigen presenting cells(APC) to allow activation of “memory” T cells, as compared to “naive” Tcells. At the end of the culture period, T cell activity is detectedusing assays including ⁵¹ Cr release involving peptide-sensitizedtargets, T cell proliferation, or lymphokine release.

VI.) 161P2F10B Transgenic Animals

Nucleic acids that encode a 161P2F10B-related protein can also be usedto generate either transgenic animals or “knock out” animals that, inturn, are useful in the development and screening of therapeuticallyuseful reagents. In accordance with established techniques, cDNAencoding 161P2F10B can be used to clone genomic DNA that encodes161P2F10B. The cloned genomic sequences can then be used to generatetransgenic animals containing cells that express DNA that encode161P2F10B. Methods for generating transgenic animals, particularlyanimals such as mice or rats, have become conventional in the art andare described, for example, in U.S. Pat. No. 4,736,866 issued 12 Apr.1988, and U.S. Pat. No. 4,870,009 issued 26 Sep. 1989. Typically,particular cells would be targeted for 161P2F10B transgene incorporationwith tissue-specific enhancers.

Transgenic animals that include a copy of a transgene encoding 161P2F10Bcan be used to examine the effect of increased expression of DNA thatencodes 161P2F10B. Such animals can be used as tester animals forreagents thought to confer protection from, for example, pathologicalconditions associated with its overexpression. In accordance with thisaspect of the invention, an animal is treated with a reagent and areduced incidence of a pathological condition, compared to untreatedanimals that bear the transgene, would indicate a potential therapeuticintervention for the pathological condition.

Alternatively, non-human homologues of 161P2F10B can be used toconstruct a 161P2F10B “knock out” animal that has a defective or alteredgene encoding 161P2F10B as a result of homologous recombination betweenthe endogenous gene encoding 161P2F10B and altered genomic DNA encoding161P2F10B introduced into an embryonic cell of the animal. For example,cDNA that encodes 161P2F10B can be used to clone genomic DNA encoding161P2F10B in accordance with established techniques. A portion of thegenomic DNA encoding 161P2F10B can be deleted or replaced with anothergene, such as a gene encoding a selectable marker that can be used tomonitor integration. Typically, several kilobases of unaltered flankingDNA (both at the 5′ and 3′ ends) are included in the vector (see, e.g.,Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected(see, e.g., Li et al., Cell, 69:915 (1992)). The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras (see, e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152). A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal, and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knock out animals can becharacterized, for example, for their ability to defend against certainpathological conditions or for their development of pathologicalconditions due to absence of a 161P2F10B polypeptide.

VII.) Methods for the Detection of 161P2F10B

Another aspect of the present invention relates to methods for detecting161P2F10B polynucleotides and 161P2F10B-related proteins, as well asmethods for identifying a cell that expresses 161P2F10B. The expressionprofile of 161P2F10B makes it a diagnostic marker for metastasizeddisease. Accordingly, the status of 161P2F10B gene products providesinformation useful for predicting a variety of factors includingsusceptibility to advanced stage disease, rate of progression, and/ortumor aggressiveness. As discussed in detail herein, the status of161P2F10B gene products in patient samples can be analyzed by a varietyprotocols that are well known in the art including immunohistochemicalanalysis, the variety of Northern blotting techniques including in situhybridization, RT-PCR analysis (for example on laser capturemicro-dissected samples), Western blot analysis and tissue arrayanalysis.

More particularly, the invention provides assays for the detection of161P2F10B polynucleotides in a biological sample, such as serum, bone,prostate, and other tissues, urine, semen, cell preparations, and thelike. Detectable 161P2F10B polynucleotides include, for example, a161P2F10B gene or fragment thereof, 161P2F10B mRNA, alternative splicevariant 161P2F10B mRNAs, and recombinant DNA or RNA molecules thatcontain a 161P2F10B polynucleotide. A number of methods for amplifyingand/or detecting the presence of 161P2F10B polynucleotides are wellknown in the art and can be employed in the practice of this aspect ofthe invention.

In one embodiment, a method for detecting a 161P2F10B mRNA in abiological sample comprises producing cDNA from the sample by reversetranscription using at least one primer; amplifying the cDNA so producedusing a 161P2F10B polynucleotides as sense and antisense primers toamplify 161P2F10B cDNAs therein; and detecting the presence of theamplified 161P2F10B cDNA. Optionally, the sequence of the amplified161P2F10B cDNA can be determined.

In another embodiment, a method of detecting a 161P2F10B gene in abiological sample comprises first isolating genomic DNA from the sample;amplifying the isolated genomic DNA using 161P2F10B polynucleotides assense and antisense primers; and detecting the presence of the amplified161P2F10B gene. Any number of appropriate sense and antisense probecombinations can be designed from a 161P2F10B nucleotide sequence (see,e.g., FIG. 2) and used for this purpose.

The invention also provides assays for detecting the presence of a161P2F10B protein in a tissue or other biological sample such as serum,semen, bone, prostate, urine, cell preparations, and the like. Methodsfor detecting a 161P2F10B-related protein are also well known andinclude, for example, immunoprecipitation, immunohistochemical analysis,Western blot analysis, molecular binding assays, ELISA, ELIFA and thelike. For example, a method of detecting the presence of a161P2F10B-related protein in a biological sample comprises firstcontacting the sample with a 161P2F10B antibody, a 161P2F10B-reactivefragment thereof, or a recombinant protein containing an antigen-bindingregion of a 161P2F10B antibody; and then detecting the binding of161P2F10B-related protein in the sample.

Methods for identifying a cell that expresses 161P2F10B are also withinthe scope of the invention. In one embodiment, an assay for identifyinga cell that expresses a 161P2F10B gene comprises detecting the presenceof 161P2F10B mRNA in the cell. Methods for the detection of particularmRNAs in cells are well known and include, for example, hybridizationassays using complementary DNA probes (such as in situ hybridizationusing labeled 161P2F10B riboprobes, Northern blot and relatedtechniques) and various nucleic acid amplification assays (such asRT-PCR using complementary primers specific for 161P2F10B, and otheramplification type detection methods, such as, for example, branchedDNA, SISBA, TMA and the like). Alternatively, an assay for identifying acell that expresses a 161P2F10B gene comprises detecting the presence of161P2F10B-related protein in the cell or secreted by the cell. Variousmethods for the detection of proteins are well known in the art and areemployed for the detection of 161P2F10B-related proteins and cells thatexpress 161P2F10B-related proteins.

161P2F10B expression analysis is also useful as a tool for identifyingand evaluating agents that modulate 161P2F10B gene expression. Forexample, 161P2F10B expression is significantly upregulated in prostatecancer, and is expressed in cancers of the tissues listed in Table I.Identification of a molecule or biological agent that inhibits 161P2F10Bexpression or over-expression in cancer cells is of therapeutic value.For example, such an agent can be identified by using a screen thatquantifies 161P2F10B expression by RT-PCR, nucleic acid hybridization orantibody binding.

VIII.) Methods for Monitoring the Status of 161P2F10B-Related Genes andTheir Products

Oncogenesis is known to be a multistep process where cellular growthbecomes progressively dysregulated and cells progress from a normalphysiological state to precancerous and then cancerous states (see,e.g., Alers et al., Lab Invest. 77(5): 437-438 (1997) and Isaacs et al.,Cancer Surv. 23: 19-32 (1995)). In this context, examining a biologicalsample for evidence of dysregulated cell growth (such as aberrant161P2F10B expression in cancers) allows for early detection of suchaberrant physiology, before a pathologic state such as cancer hasprogressed to a stage that therapeutic options are more limited and orthe prognosis is worse. In such examinations, the status of 161P2F10B ina biological sample of interest can be compared, for example, to thestatus of 161P2F10B in a corresponding normal sample (e.g. a sample fromthat individual or alternatively another individual that is not affectedby a pathology). An alteration in the status of 161P2F10B in thebiological sample (as compared to the normal sample) provides evidenceof dysregulated cellular growth. In addition to using a biologicalsample that is not affected by a pathology as a normal sample, one canalso use a predetermined normative value such as a predetermined normallevel of mRNA expression (see, e.g., Greyer et al., J. Comp. Neurol.1996 Dec. 9; 376(2): 306-14 and U.S. Pat. No. 5,837,501) to compare161P2F10B status in a sample.

The term “status” in this context is used according to its art acceptedmeaning and refers to the condition or state of a gene and its products.Typically, skilled artisans use a number of parameters to evaluate thecondition or state of a gene and its products. These include, but arenot limited to the location of expressed gene products (including thelocation of 161P2F10B expressing cells) as well as the level, andbiological activity of expressed gene products (such as 161P2F10B mRNA,polynucleotides and polypeptides). Typically, an alteration in thestatus of 161P2F10B comprises a change in the location of 161P2F10Band/or 161P2F10B expressing cells and/or an increase in 161P2F10B mRNAand/or protein expression.

161P2F10B status in a sample can be analyzed by a number of means wellknown in the art, including without limitation, immunohistochemicalanalysis, in situ hybridization, RT-PCR analysis on laser capturemicro-dissected samples, Western blot analysis, and tissue arrayanalysis. Typical protocols for evaluating the status of a 161P2F10Bgene and gene products are found, for example in Ausubel et al. eds.,1995, Current Protocols In Molecular Biology, Units 2 (NorthernBlotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCRAnalysis). Thus, the status of 161P2F10B in a biological sample isevaluated by various methods utilized by skilled artisans including, butnot limited to genomic Southern analysis (to examine, for exampleperturbations in a 161P2F10B gene), Northern analysis and/or PCRanalysis of 161P2F10B mRNA (to examine, for example alterations in thepolynucleotide sequences or expression levels of 161P2F10B mRNAs), and,Western and/or immunohistochemical analysis (to examine, for examplealterations in polypeptide sequences, alterations in polypeptidelocalization within a sample, alterations in expression levels of161P2F10B proteins and/or associations of 161P2F10B proteins withpolypeptide binding partners). Detectable 161P2F10B polynucleotidesinclude, for example, a 161P2F10B gene or fragment thereof, 161P2F10BmRNA, alternative splice variants, 161P2F10B mRNAs, and recombinant DNAor RNA molecules containing a 161P2F10B polynucleotide.

The expression profile of 161P2F10B makes it a diagnostic marker forlocal and/or metastasized disease, and provides information on thegrowth or oncogenic potential of a biological sample. In particular, thestatus of 161P2F10B provides information useful for predictingsusceptibility to particular disease stages, progression, and/or tumoraggressiveness. The invention provides methods and assays fordetermining 161P2F10B status and diagnosing cancers that express161P2F10B, such as cancers of the tissues listed in Table I. Forexample, because 161P2F10B mRNA is so highly expressed in prostate andother cancers relative to normal prostate tissue, assays that evaluatethe levels of 161P2F10B mRNA transcripts or proteins in a biologicalsample can be used to diagnose a disease associated with 161P2F10Bdysregulation, and can provide prognostic information useful in definingappropriate therapeutic options.

The expression status of 161P2F10B provides information including thepresence, stage and location of dysplastic, precancerous and cancerouscells, predicting susceptibility to various stages of disease, and/orfor gauging tumor aggressiveness. Moreover, the expression profile makesit useful as an imaging reagent for metastasized disease. Consequently,an aspect of the invention is directed to the various molecularprognostic and diagnostic methods for examining the status of 161P2F10Bin biological samples such as those from individuals suffering from, orsuspected of suffering from a pathology characterized by dysregulatedcellular growth, such as cancer.

As described above, the status of 161P2F10B in a biological sample canbe examined by a number of well-known procedures in the art. Forexample, the status of 161P2F10B in a biological sample taken from aspecific location in the body can be examined by evaluating the samplefor the presence or absence of 161P2F10B expressing cells (e.g. thosethat express 161P2F10B mRNAs or proteins). This examination can provideevidence of dysregulated cellular growth, for example, when161P2F10B-expressing cells are found in a biological sample that doesnot normally contain such cells (such as a lymph node), because suchalterations in the status of 161P2F10B in a biological sample are oftenassociated with dysregulated cellular growth. Specifically, oneindicator of dysregulated cellular growth is the metastases of cancercells from an organ of origin (such as the prostate) to a different areaof the body (such as a lymph node). In this context, evidence ofdysregulated cellular growth is important for example because occultlymph node metastases can be detected in a substantial proportion ofpatients with prostate cancer, and such metastases are associated withknown predictors of disease progression (see, e.g., Murphy et al.,Prostate 42(4): 315-317 (2000); Su et al., Semin Surg. Oncol. 18(1):17-28 (2000) and Freeman et al., J Urol 1995 August 154(2 Pt 1):474-8).

In one aspect, the invention provides methods for monitoring 161P2F10Bgene products by determining the status of 161P2F10B gene productsexpressed by cells from an individual suspected of having a diseaseassociated with dysregulated cell growth (such as hyperplasia or cancer)and then comparing the status so determined to the status of 161P2F10Bgene products in a corresponding normal sample. The presence of aberrant161P2F10B gene products in the test sample relative to the normal sampleprovides an indication of the presence of dysregulated cell growthwithin the cells of the individual.

In another aspect, the invention provides assays useful in determiningthe presence of cancer in an individual, comprising detecting asignificant increase in 161P2F10B mRNA or protein expression in a testcell or tissue sample relative to expression levels in the correspondingnormal cell or tissue. The presence of 161P2F10B mRNA can, for example,be evaluated in tissues including but not limited to those listed inTable I. The presence of significant 161P2F10B expression in any ofthese tissues is useful to indicate the emergence, presence and/orseverity of a cancer, since the corresponding normal tissues do notexpress 161P2F10B mRNA or express it at lower levels.

In a related embodiment, 161P2F10B status is determined at the proteinlevel rather than at the nucleic acid level. For example, such a methodcomprises determining the level of 161P2F10B protein expressed by cellsin a test tissue sample and comparing the level so determined to thelevel of 161P2F10B expressed in a corresponding normal sample. In oneembodiment, the presence of 161P2F10B protein is evaluated, for example,using immunohistochemical methods. 161P2F10B antibodies or bindingpartners capable of detecting 161P2F10B protein expression are used in avariety of assay formats well known in the art for this purpose.

In a further embodiment, one can evaluate the status of 161P2F10Bnucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules. Theseperturbations can include insertions, deletions, substitutions and thelike. Such evaluations are useful because perturbations in thenucleotide and amino acid sequences are observed in a large number ofproteins associated with a growth dysregulated phenotype (see, e.g.,Marrogi et al., 1999, J. Cutan. Pathol. 26(8):369-378). For example, amutation in the sequence of 161P2F10B may be indicative of the presenceor promotion of a tumor. Such assays therefore have diagnostic andpredictive value where a mutation in 161P2F10B indicates a potentialloss of function or increase in tumor growth.

A wide variety of assays for observing perturbations in nucleotide andamino acid sequences are well known in the art. For example, the sizeand structure of nucleic acid or amino acid sequences of 161P2F10B geneproducts are observed by the Northern, Southern, Western, PCR and DNAsequencing protocols discussed herein. In addition, other methods forobserving perturbations in nucleotide and amino acid sequences such assingle strand conformation polymorphism analysis are well known in theart (see, e.g., U.S. Pat. No. 5,382,510 issued 7 Sep. 1999, and U.S.Pat. No. 5,952,170 issued 17 Jan. 1995).

Additionally, one can examine the methylation status of a 161P2F10B genein a biological sample. Aberrant demethylation and/or hypermethylationof CpG islands in gene 5′ regulatory regions frequently occurs inimmortalized and transformed cells, and can result in altered expressionof various genes. For example, promoter hypermethylation of the pi-classglutathione S-transferase (a protein expressed in normal prostate butnot expressed in >90% of prostate carcinomas) appears to permanentlysilence transcription of this gene and is the most frequently detectedgenomic alteration in prostate carcinomas (De Marzo et al., Am. J.Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration ispresent in at least 70% of cases of high-grade prostatic intraepithelialneoplasia (PIN) (Brooks et al., Cancer Epidemiol. Biomarkers Prey.,1998, 7:531-536). In another example, expression of the LAGE-I tumorspecific gene (which is not expressed in normal prostate but isexpressed in 25-50% of prostate cancers) is induced by deoxy-azacytidinein lymphoblastoid cells, suggesting that tumoral expression is due todemethylation (Lethe et al., Int. J. Cancer 76(6): 903-908 (1998)). Avariety of assays for examining methylation status of a gene are wellknown in the art. For example, one can utilize, in Southernhybridization approaches, methylation-sensitive restriction enzymes thatcannot cleave sequences that contain methylated CpG sites to assess themethylation status of CpG islands. In addition, MSP (methylationspecific PCR) can rapidly profile the methylation status of all the CpGsites present in a CpG island of a given gene. This procedure involvesinitial modification of DNA by sodium bisulfite (which will convert allunmethylated cytosines to uracil) followed by amplification usingprimers specific for methylated versus unmethylated DNA. Protocolsinvolving methylation interference can also be found for example inCurrent Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel etal. eds., 1995.

Gene amplification is an additional method for assessing the status of161P2F10B. Gene amplification is measured in a sample directly, forexample, by conventional Southern blotting or Northern blotting toquantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad.Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies are employed thatrecognize specific duplexes, including DNA duplexes, RNA duplexes, andDNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turnare labeled and the assay carried out where the duplex is bound to asurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

Biopsied tissue or peripheral blood can be conveniently assayed for thepresence of cancer cells using for example, Northern, dot blot or RT-PCRanalysis to detect 161P2F10B expression. The presence of RT-PCRamplifiable 161P2F10B mRNA provides an indication of the presence ofcancer. RT-PCR assays are well known in the art. RT-PCR detection assaysfor tumor cells in peripheral blood are currently being evaluated foruse in the diagnosis and management of a number of human solid tumors.In the prostate cancer field, these include RT-PCR assays for thedetection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol.Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13:1195-2000;Heston et al., 1995, Clin. Chem. 41:1687-1688).

A further aspect of the invention is an assessment of the susceptibilitythat an individual has for developing cancer. In one embodiment, amethod for predicting susceptibility to cancer comprises detecting161P2F10B mRNA or 161P2F10B protein in a tissue sample, its presenceindicating susceptibility to cancer, wherein the degree of 161P2F10BmRNA expression correlates to the degree of susceptibility. In aspecific embodiment, the presence of 161P2F10B in prostate or othertissue is examined, with the presence of 161P2F10B in the sampleproviding an indication of prostate cancer susceptibility (or theemergence or existence of a prostate tumor). Similarly, one can evaluatethe integrity 161P2F10B nucleotide and amino acid sequences in abiological sample, in order to identify perturbations in the structureof these molecules such as insertions, deletions, substitutions and thelike. The presence of one or more perturbations in 161P2F10B geneproducts in the sample is an indication of cancer susceptibility (or theemergence or existence of a tumor).

The invention also comprises methods for gauging tumor aggressiveness.In one embodiment, a method for gauging aggressiveness of a tumorcomprises determining the level of 161P2F10B mRNA or 161P2F10B proteinexpressed by tumor cells, comparing the level so determined to the levelof 161P2F10B mRNA or 161P2F10B protein expressed in a correspondingnormal tissue taken from the same individual or a normal tissuereference sample, wherein the degree of 161P2F10B mRNA or 161P2F10Bprotein expression in the tumor sample relative to the normal sampleindicates the degree of aggressiveness. In a specific embodiment,aggressiveness of a tumor is evaluated by determining the extent towhich 161P2F10B is expressed in the tumor cells, with higher expressionlevels indicating more aggressive tumors. Another embodiment is theevaluation of the integrity of 161P2F10B nucleotide and amino acidsequences in a biological sample, in order to identify perturbations inthe structure of these molecules such as insertions, deletions,substitutions and the like. The presence of one or more perturbationsindicates more aggressive tumors.

Another embodiment of the invention is directed to methods for observingthe progression of a malignancy in an individual over time. In oneembodiment, methods for observing the progression of a malignancy in anindividual over time comprise determining the level of 161P2F10B mRNA or161P2F10B protein expressed by cells in a sample of the tumor, comparingthe level so determined to the level of 161P2F10B mRNA or 161P2F10Bprotein expressed in an equivalent tissue sample taken from the sameindividual at a different time, wherein the degree of 161P2F10B mRNA or161P2F10B protein expression in the tumor sample over time providesinformation on the progression of the cancer. In a specific embodiment,the progression of a cancer is evaluated by determining 161P2F10Bexpression in the tumor cells over time, where increased expression overtime indicates a progression of the cancer. Also, one can evaluate theintegrity 161P2F10B nucleotide and amino acid sequences in a biologicalsample in order to identify perturbations in the structure of thesemolecules such as insertions, deletions, substitutions and the like,where the presence of one or more perturbations indicates a progressionof the cancer.

The above diagnostic approaches can be combined with any one of a widevariety of prognostic and diagnostic protocols known in the art. Forexample, another embodiment of the invention is directed to methods forobserving a coincidence between the expression of 161P2F10B gene and161P2F10B gene products (or perturbations in 161P2F10B gene and161P2F10B gene products) and a factor that is associated withmalignancy, as a means for diagnosing and prognosticating the status ofa tissue sample. A wide variety of factors associated with malignancycan be utilized, such as the expression of genes associated withmalignancy (e.g. PSA, PSCA and PSM expression for prostate cancer etc.)as well as gross cytological observations (see, e.g., Bocking et al.,1984, Anal. Quant. Cytol. 6(2):74-88; Epstein, 1995, Hum. Pathol.26(2):223-9; Thorson et al., 1998, Mod. Pathol. 11(6):543-51; Baisden etal., 1999, Am. J. Surg. Pathol. 23(8):918-24). Methods for observing acoincidence between the expression of 161P2F10B gene and 161P2F10B geneproducts (or perturbations in 161P2F10B gene and 161P2F10B geneproducts) and another factor that is associated with malignancy areuseful, for example, because the presence of a set of specific factorsthat coincide with disease provides information crucial for diagnosingand prognosticating the status of a tissue sample.

In one embodiment, methods for observing a coincidence between theexpression of 161P2F10B gene and 161P2F10B gene products (orperturbations in 161P2F10B gene and 161P2F10B gene products) and anotherfactor associated with malignancy entails detecting the overexpressionof 161P2F10B mRNA or protein in a tissue sample, detecting theoverexpression of PSA mRNA or protein in a tissue sample (or PSCA or PSMexpression), and observing a coincidence of 161P2F10B mRNA or proteinand PSA mRNA or protein overexpression (or PSCA or PSM expression). In aspecific embodiment, the expression of 161P2F10B and PSA mRNA inprostate tissue is examined, where the coincidence of 161P2F10B and PSAmRNA overexpression in the sample indicates the existence of prostatecancer, prostate cancer susceptibility or the emergence or status of aprostate tumor.

Methods for detecting and quantifying the expression of 161P2F10B mRNAor protein are described herein, and standard nucleic acid and proteindetection and quantification technologies are well known in the art.Standard methods for the detection and quantification of 161P2F10B mRNAinclude in situ hybridization using labeled 161P2F10B riboprobes,Northern blot and related techniques using 161P2F10B polynucleotideprobes, RT-PCR analysis using primers specific for 161P2F10B, and otheramplification type detection methods, such as, for example, branchedDNA, SISBA, TMA and the like. In a specific embodiment,semi-quantitative RT-PCR is used to detect and quantify 161P2F10B mRNAexpression. Any number of primers capable of amplifying 161P2F10B can beused for this purpose, including but not limited to the various primersets specifically described herein. In a specific embodiment, polyclonalor monoclonal antibodies specifically reactive with the wild-type161P2F10B protein can be used in an immunohistochemical assay ofbiopsied tissue.

IX.) Identification of Molecules That Interact With 161P2F10B

The 161P2F10B protein and nucleic acid sequences disclosed herein allowa skilled artisan to identify proteins, small molecules and other agentsthat interact with 161P2F10B, as well as pathways activated by 161P2F10Bvia any one of a variety of art accepted protocols. For example, one canutilize one of the so-called interaction trap systems (also referred toas the “two-hybrid assay”). In such systems, molecules interact andreconstitute a transcription factor which directs expression of areporter gene, whereupon the expression of the reporter gene is assayed.Other systems identify protein-protein interactions in vivo throughreconstitution of a eukaryotic transcriptional activator, see, e.g.,U.S. Pat. No. 5,955,280 issued 21 Sep. 1999, U.S. Pat. No. 5,925,523issued 20 Jul. 1999, U.S. Pat. No. 5,846,722 issued 8 Dec. 1998 and U.S.Pat. No. 6,004,746 issued 21 Dec. 1999. Algorithms are also available inthe art for genome-based predictions of protein function (see, e.g.,Marcotte, et al., Nature 402: 4 Nov. 1999, 83-86).

Alternatively one can screen peptide libraries to identify moleculesthat interact with 161P2F10B protein sequences. In such methods,peptides that bind to 161P2F10B are identified by screening librariesthat encode a random or controlled collection of amino acids. Peptidesencoded by the libraries are expressed as fusion proteins ofbacteriophage coat proteins, the bacteriophage particles are thenscreened against the 161P2F10B protein(s).

Accordingly, peptides having a wide variety of uses, such astherapeutic, prognostic or diagnostic reagents, are thus identifiedwithout any prior information on the structure of the expected ligand orreceptor molecule. Typical peptide libraries and screening methods thatcan be used to identify molecules that interact with 161P2F10B proteinsequences are disclosed for example in U.S. Pat. No. 5,723,286 issued 3Mar. 1998 and U.S. Pat. No. 5,733,731 issued 31 Mar. 1998.

Alternatively, cell lines that express 161P2F10B are used to identifyprotein-protein interactions mediated by 161P2F10B. Such interactionscan be examined using immunoprecipitation techniques (see, e.g.,Hamilton B. J., et al. Biochem. Biophys. Res. Commun 1999, 261:646-51).161P2F10B protein can be immunoprecipitated from 161P2F10B-expressingcell lines using anti-161P2F10B antibodies. Alternatively, antibodiesagainst His-tag can be used in a cell line engineered to express fusionsof 161P2F10B and a His-tag (vectors mentioned above). Theimmunoprecipitated complex can be examined for protein association byprocedures such as Western blotting, 35S-methionine labeling ofproteins, protein microsequencing, silver staining and two-dimensionalgel electrophoresis.

Small molecules and ligands that interact with 161P2F10B can beidentified through related embodiments of such screening assays. Forexample, small molecules can be identified that interfere with proteinfunction, including molecules that interfere with 161P2F10B's ability tomediate phosphorylation and de-phosphorylation, interaction with DNA orRNA molecules as an indication of regulation of cell cycles, secondmessenger signaling or tumorigenesis. Similarly, small molecules thatmodulate 161P2F10B-related ion channel, protein pump, or cellcommunication functions are identified and used to treat patients thathave a cancer that expresses 161P2F10B (see, e.g., Hille, B., IonicChannels of Excitable Membranes 2nd Ed., Sinauer Assoc., Sunderland,Mass., 1992). Moreover, ligands that regulate 161P2F10B function can beidentified based on their ability to bind 161P2F10B and activate areporter construct. Typical methods are discussed for example in U.S.Pat. No. 5,928,868 issued 27 Jul. 1999, and include methods for forminghybrid ligands in which at least one ligand is a small molecule. In anillustrative embodiment, cells engineered to express a fusion protein of161P2F10B and a DNA-binding protein are used to co-express a fusionprotein of a hybrid ligand/small molecule and a cDNA librarytranscriptional activator protein. The cells further contain a reportergene, the expression of which is conditioned on the proximity of thefirst and second fusion proteins to each other, an event that occursonly if the hybrid ligand binds to target sites on both hybrid proteins.Those cells that express the reporter gene are selected and the unknownsmall molecule or the unknown ligand is identified. This method providesa means of identifying modulators, which activate or inhibit 161P2F10B.

An embodiment of this invention comprises a method of screening for amolecule that interacts with a 161P2F10B amino acid sequence shown inFIG. 2 or FIG. 3, comprising the steps of contacting a population ofmolecules with a 161P2F10B amino acid sequence, allowing the populationof molecules and the 161P2F10B amino acid sequence to interact underconditions that facilitate an interaction, determining the presence of amolecule that interacts with the 161P2F10B amino acid sequence, and thenseparating molecules that do not interact with the 161P2F10B amino acidsequence from molecules that do. In a specific embodiment, the methodfurther comprises purifying, characterizing and identifying a moleculethat interacts with the 161P2F10B amino acid sequence. The identifiedmolecule can be used to modulate a function performed by 161P2F10B. In apreferred embodiment, the 161P2F10B amino acid sequence is contactedwith a library of peptides.

X.) Therapeutic Methods and Compositions

The identification of 161P2F10B as a protein that is normally expressedin a restricted set of tissues, but which is also expressed in prostateand other cancers, opens a number of therapeutic approaches to thetreatment of such cancers. As contemplated herein, 161P2F10B functionsas a transcription factor involved in activating tumor-promoting genesor repressing genes that block tumorigenesis.

Accordingly, therapeutic approaches that inhibit the activity of a161P2F10B protein are useful for patients suffering from a cancer thatexpresses 161P2F10B. These therapeutic approaches generally fall intotwo classes. One class comprises various methods for inhibiting thebinding or association of a 161P2F10B protein with its binding partneror with other proteins. Another class comprises a variety of methods forinhibiting the transcription of a 161P2F10B gene or translation of161P2F10B mRNA.

X.A.) Anti-Cancer Vaccines

The invention provides cancer vaccines comprising a 161P2F10B-relatedprotein or 161P2F10B-related nucleic acid. In view of the expression of161P2F10B, cancer vaccines prevent and/or treat 161P2F10B-expressingcancers with minimal or no effects on non-target tissues. The use of atumor antigen in a vaccine that generates humoral and/or cell-mediatedimmune responses as anti-cancer therapy is well known in the art and hasbeen employed in prostate cancer using human PSMA and rodent PAPimmunogens (Hodge et al., 1995, Int. J. Cancer 63:231-237; Fong et al.,1997, J. Immunol. 159:3113-3117).

Such methods can be readily practiced by employing a 161P2F10B-relatedprotein, or a 161P2F10B-encoding nucleic acid molecule and recombinantvectors capable of expressing and presenting the 161P2F10B immunogen(which typically comprises a number of antibody or T cell epitopes).Skilled artisans understand that a wide variety of vaccine systems fordelivery of immunoreactive epitopes are known in the art (see, e.g.,Heryln et al., Ann Med 1999 Feb. 31(1):66-78; Maruyama et al., CancerImmunol Immunother 2000 Jun. 49(3):123-32) Briefly, such methods ofgenerating an immune response (e.g. humoral and/or cell-mediated) in amammal, comprise the steps of: exposing the mammal's immune system to animmunoreactive epitope (e.g. an epitope present in a 161P2F10B proteinshown in FIG. 3 or analog or homolog thereof) so that the mammalgenerates an immune response that is specific for that epitope (e.g.generates antibodies that specifically recognize that epitope). In apreferred method, a 161P2F10B immunogen contains a biological motif, seee.g., Tables VIII-XXI and XXII-XLIX, or a peptide of a size range from161P2F10B indicated in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9.

The entire 161P2F10B protein, immunogenic regions or epitopes thereofcan be combined and delivered by various means. Such vaccinecompositions can include, for example, lipopeptides (e.g., Vitiello, A.et al., J. Clin. Invest. 95:341, 1995), peptide compositionsencapsulated in poly(DL-lactide-co-glycolide) (“PLG”) microspheres (see,e.g., Eldridge, et al., Molec. Immunol. 28:287-294, 1991: Alonso et al.,Vaccine 12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995),peptide compositions contained in immune stimulating complexes (ISCOMS)(see, e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., ClinExp Immunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs)(see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988;Tam, J. P., J. Immunol. Methods 196:17-32, 1996), peptides formulated asmultivalent peptides; peptides for use in ballistic delivery systems,typically crystallized peptides, viral delivery vectors (Perkus, M. E.et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p.379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. etal., Nature 320:537, 1986; Kieny, M.-P. et al., AIDS Bio/Technology4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda,P. K. et al., Virology 175:535, 1990), particles of viral or syntheticorigin (e.g., Kofler, N. et al., J. Immunol. Methods. 192:25, 1996;Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. etal., Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R.,and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al.,Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol.148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked orparticle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993;Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993;Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S.H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev.Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16,1993). Toxin-targeted delivery technologies, also known as receptormediated targeting, such as those of Avant Immunotherapeutics, Inc.(Needham, Mass.) may also be used.

In patients with 161P2F10B-associated cancer, the vaccine compositionsof the invention can also be used in conjunction with other treatmentsused for cancer, e.g., surgery, chemotherapy, drug therapies, radiationtherapies, etc. including use in combination with immune adjuvants suchas IL-2, IL-12, GM-CSF, and the like.

Cellular Vaccines:

CTL epitopes can be determined using specific algorithms to identifypeptides within 161P2F10B protein that bind corresponding HLA alleles(see e.g., Table IV; Epimer™ and Epimatrix™, BIMAS, and SYFPEITHI). In apreferred embodiment, a 161P2F10B immunogen contains one or more aminoacid sequences identified using techniques well known in the art, suchas the sequences shown in Tables VIII-XXI and XXII-XLIX or a peptide of8, 9, 10 or 11 amino acids specified by an HLA Class I motif/supermotif(e.g., Table IV (A), Table IV (D), or Table IV (E)) and/or a peptide ofat least 9 amino acids that comprises an HLA Class II motif/supermotif(e.g., Table IV (B) or Table IV (C)). As is appreciated in the art, theHLA Class I binding groove is essentially closed ended so that peptidesof only a particular size range can fit into the groove and be bound,generally HLA Class I epitopes are 8, 9, 10, or 11 amino acids long. Incontrast, the HLA Class II binding groove is essentially open ended;therefore a peptide of about 9 or more amino acids can be bound by anHLA Class II molecule. Due to the binding groove differences between HLAClass I and II, HLA Class I motifs are length specific, i.e., positiontwo of a Class I motif is the second amino acid in an amino to carboxyldirection of the peptide. The amino acid positions in a Class II motifare relative only to each other, not the overall peptide, i.e.,additional amino acids can be attached to the amino and/or carboxyltermini of a motif-bearing sequence. HLA Class II epitopes are often 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 aminoacids long, or longer than 25 amino acids.

Antibody-Based Vaccines

A wide variety of methods for generating an immune response in a mammalare known in the art (for example as the first step in the generation ofhybridomas). Methods of generating an immune response in a mammalcomprise exposing the mammal's immune system to an immunogenic epitopeon a protein (e.g. a 161P2F10B protein) so that an immune response isgenerated. A typical embodiment consists of a method for generating animmune response to 161P2F10B in a host, by contacting the host with asufficient amount of at least one 161P2F10B B cell or cytotoxic T-cellepitope or analog thereof; and at least one periodic interval thereafterre-contacting the host with the 161P2F10B B cell or cytotoxic T-cellepitope or analog thereof. A specific embodiment consists of a method ofgenerating an immune response against a 161P2F10B-related protein or aman-made multiepitopic peptide comprising: administering 161P2F10Bimmunogen (e.g. a 161P2F10B protein or a peptide fragment thereof, a161P2F10B fusion protein or analog etc.) in a vaccine preparation to ahuman or another mammal. Typically, such vaccine preparations furthercontain a suitable adjuvant (see, e.g., U.S. Pat. No. 6,146,635) or auniversal helper epitope such as a PADRE™ peptide (Epimmune Inc., SanDiego, Calif.; see, e.g., Alexander et al., J. Immunol. 2000 164(3);164(3): 1625-1633; Alexander et al., Immunity 1994 1(9): 751-761 andAlexander et al., Immunol. Res. 1998 18(2): 79-92). An alternativemethod comprises generating an immune response in an individual againsta 161P2F10B immunogen by: administering in vivo to muscle or skin of theindividual's body a DNA molecule that comprises a DNA sequence thatencodes a 161P2F10B immunogen, the DNA sequence operatively linked toregulatory sequences which control the expression of the DNA sequence;wherein the DNA molecule is taken up by cells, the DNA sequence isexpressed in the cells and an immune response is generated against theimmunogen (see, e.g., U.S. Pat. No. 5,962,428). Optionally a geneticvaccine facilitator such as anionic lipids; saponins; lectins;estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; andurea is also administered. In addition, an antiidiotypic antibody can beadministered that mimics 161P2F10B, in order to generate a response tothe target antigen.

Nucleic Acid Vaccines:

Vaccine compositions of the invention include nucleic acid-mediatedmodalities. DNA or RNA that encode protein(s) of the invention can beadministered to a patient. Genetic immunization methods can be employedto generate prophylactic or therapeutic humoral and cellular immuneresponses directed against cancer cells expressing 161P2F10B. Constructscomprising DNA encoding a 161P2F10B-related protein/immunogen andappropriate regulatory sequences can be injected directly into muscle orskin of an individual, such that the cells of the muscle or skin take-upthe construct and express the encoded 161P2F10B protein/immunogen.Alternatively, a vaccine comprises a 161P2F10B-related protein.Expression of the 161P2F10B-related protein immunogen results in thegeneration of prophylactic or therapeutic humoral and cellular immunityagainst cells that bear a 161P2F10B protein. Various prophylactic andtherapeutic genetic immunization techniques known in the art can be used(for review, see information and references published at Internetaddress genweb.com). Nucleic acid-based delivery is described, forinstance, in Wolff et. al., Science 247:1465 (1990) as well as U.S. Pat.Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647;WO 98/04720. Examples of DNA-based delivery technologies include “nakedDNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery,cationic lipid complexes, and particle-mediated (“gene gun”) orpressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687).

For therapeutic or prophylactic immunization purposes, proteins of theinvention can be expressed via viral or bacterial vectors. Various viralgene delivery systems that can be used in the practice of the inventioninclude, but are not limited to, vaccinia, fowlpox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and sindbis virus (see, e.g., Restifo, 1996, Curr. Opin. Immunol.8:658-663; Tsang et al. J. Natl. Cancer Inst. 87:982-990 (1995)).Non-viral delivery systems can also be employed by introducing naked DNAencoding a 161P2F10B-related protein into the patient (e.g.,intramuscularly or intradermally) to induce an anti-tumor response.

Vaccinia virus is used, for example, as a vector to express nucleotidesequences that encode the peptides of the invention. Upon introductioninto a host, the recombinant vaccinia virus expresses the proteinimmunogenic peptide, and thereby elicits a host immune response.Vaccinia vectors and methods useful in immunization protocols aredescribed in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG(Bacille Calmette Guerin). BCG vectors are described in Stover et al.,Nature 351:456-460 (1991). A wide variety of other vectors useful fortherapeutic administration or immunization of the peptides of theinvention, e.g. adeno and adeno-associated virus vectors, retroviralvectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, andthe like, will be apparent to those skilled in the art from thedescription herein.

Thus, gene delivery systems are used to deliver a 161P2F10B-relatednucleic acid molecule. In one embodiment, the full-length human161P2F10B cDNA is employed. In another embodiment, 161P2F10B nucleicacid molecules encoding specific cytotoxic T lymphocyte (CTL) and/orantibody epitopes are employed.

Ex Vivo Vaccines

Various ex vivo strategies can also be employed to generate an immuneresponse. One approach involves the use of antigen presenting cells(APCs) such as dendritic cells (DC) to present 161P2F10B antigen to apatient's immune system. Dendritic cells express MHC class I and IImolecules, B7 co-stimulator, and IL-12, and are thus highly specializedantigen presenting cells. In prostate cancer, autologous dendritic cellspulsed with peptides of the prostate-specific membrane antigen (PSMA)are being used in a Phase I clinical trial to stimulate prostate cancerpatients' immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphyet al., 1996, Prostate 29:371-380). Thus, dendritic cells can be used topresent 161P2F10B peptides to T cells in the context of MHC class I orII molecules. In one embodiment, autologous dendritic cells are pulsedwith 161P2F10B peptides capable of binding to MHC class I and/or classII molecules. In another embodiment, dendritic cells are pulsed with thecomplete 161P2F10B protein. Yet another embodiment involves engineeringthe overexpression of a 161P2F10B gene in dendritic cells using variousimplementing vectors known in the art, such as adenovirus (Arthur etal., 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et al.,1996, Cancer Res. 56:3763-3770), lentivirus, adeno-associated virus, DNAtransfection (Ribas et al., 1997, Cancer Res. 57:2865-2869), ortumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med.186:1177-1182). Cells that express 161P2F10B can also be engineered toexpress immune modulators, such as GM-CSF, and used as immunizingagents.

X.B.) 161P2F10B as a Target for Antibody-Based Therapy

161P2F10B is an attractive target for antibody-based therapeuticstrategies. A number of antibody strategies are known in the art fortargeting both extracellular and intracellular molecules (see, e.g.,complement and ADCC mediated killing as well as the use of intrabodies).Because 161P2F10B is expressed by cancer cells of various lineagesrelative to corresponding normal cells, systemic administration of161P2F10B-immunoreactive compositions are prepared that exhibitexcellent sensitivity without toxic, non-specific and/or non-targeteffects caused by binding of the immunoreactive composition tonon-target organs and tissues. Antibodies specifically reactive withdomains of 161P2F10B are useful to treat 161P2F10B-expressing cancerssystemically, either as conjugates with a toxin or therapeutic agent, oras naked antibodies capable of inhibiting cell proliferation orfunction.

161P2F10B antibodies can be introduced into a patient such that theantibody binds to 161P2F10B and modulates a function, such as aninteraction with a binding partner, and consequently mediatesdestruction of the tumor cells and/or inhibits the growth of the tumorcells. Mechanisms by which such antibodies exert a therapeutic effectcan include complement-mediated cytolysis, antibody-dependent cellularcytotoxicity, modulation of the physiological function of 161P2F10B,inhibition of ligand binding or signal transduction pathways, modulationof tumor cell differentiation, alteration of tumor angiogenesis factorprofiles, and/or apoptosis.

Those skilled in the art understand that antibodies can be used tospecifically target and bind immunogenic molecules such as animmunogenic region of a 161P2F10B sequence shown in FIG. 2 or FIG. 3. Inaddition, skilled artisans understand that it is routine to conjugateantibodies to cytotoxic agents (see, e.g., Slevers et al. Blood 93:113678-3684 (Jun. 1, 1999)). When cytotoxic and/or therapeutic agents aredelivered directly to cells, such as by conjugating them to antibodiesspecific for a molecule expressed by that cell (e.g. 161P2F10B), thecytotoxic agent will exert its known biological effect (i.e.cytotoxicity) on those cells.

A wide variety of compositions and methods for using antibody-cytotoxicagent conjugates to kill cells are known in the art. In the context ofcancers, typical methods entail administering to an animal having atumor a biologically effective amount of a conjugate comprising aselected cytotoxic and/or therapeutic agent linked to a targeting agent(e.g. an anti-161P2F10B antibody) that binds to a marker (e.g.161P2F10B) expressed, accessible to binding or localized on the cellsurfaces. A typical embodiment is a method of delivering a cytotoxicand/or therapeutic agent to a cell expressing 161P2F10B, comprisingconjugating the cytotoxic agent to an antibody that immunospecificallybinds to a 161P2F10B epitope, and, exposing the cell to theantibody-agent conjugate. Another illustrative embodiment is a method oftreating an individual suspected of suffering from metastasized cancer,comprising a step of administering parenterally to said individual apharmaceutical composition comprising a therapeutically effective amountof an antibody conjugated to a cytotoxic and/or therapeutic agent.

Cancer immunotherapy using anti-161P2F10B antibodies can be done inaccordance with various approaches that have been successfully employedin the treatment of other types of cancer, including but not limited tocolon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133-138),multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari etal., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992,Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J.Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al.,1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al., 1994,Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res.55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin.Immunol. 11:117-127). Some therapeutic approaches involve conjugation ofnaked antibody to a toxin or radioisotope, such as the conjugation ofY91 or I131 to anti-CD20 antibodies (e.g., Zevalin™, IDECPharmaceuticals Corp. or Bexxar™, Coulter Pharmaceuticals), while othersinvolve co-administration of antibodies and other therapeutic agents,such as Herceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). Theantibodies can be conjugated to a therapeutic agent. To treat prostatecancer, for example, 161P2F10B antibodies can be administered inconjunction with radiation, chemotherapy or hormone ablation. Also,antibodies can be conjugated to a toxin such as calicheamicin (e.g.,Mylotarg™, Wyeth-Ayerst, Madison, N.J., a recombinant humanized IgG4kappa antibody conjugated to antitumor antibiotic calicheamicin) or amaytansinoid (e.g., taxane-based Tumor-Activated Prodrug, TAP, platform,ImmunoGen, Cambridge, Mass., also see e.g., U.S. Pat. No. 5,416,064).

Although 161P2F10B antibody therapy is useful for all stages of cancer,antibody therapy can be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionis indicated for patients who have received one or more rounds ofchemotherapy. Alternatively, antibody therapy of the invention iscombined with a chemotherapeutic or radiation regimen for patients whohave not received chemotherapeutic treatment. Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well. Fan et al. (Cancer Res.53:4637-4642, 1993), Prewett et al. (International J. of Onco.9:217-224, 1996), and Hancock et al. (Cancer Res. 51:4575-4580, 1991)describe the use of various antibodies together with chemotherapeuticagents.

Although 161P2F10B antibody therapy is useful for all stages of cancer,antibody therapy can be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionis indicated for patients who have received one or more rounds ofchemotherapy. Alternatively, antibody therapy of the invention iscombined with a chemotherapeutic or radiation regimen for patients whohave not received chemotherapeutic treatment. Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well.

Cancer patients can be evaluated for the presence and level of 161P2F10Bexpression, preferably using immunohistochemical assessments of tumortissue, quantitative 161P2F10B imaging, or other techniques thatreliably indicate the presence and degree of 161P2F10B expressionImmunohistochemical analysis of tumor biopsies or surgical specimens ispreferred for this purpose. Methods for immunohistochemical analysis oftumor tissues are well known in the art.

Anti-161P2F10B monoclonal antibodies that treat prostate and othercancers include those that initiate a potent immune response against thetumor or those that are directly cytotoxic. In this regard,anti-161P2F10B monoclonal antibodies (mAbs) can elicit tumor cell lysisby either complement-mediated or antibody-dependent cell cytotoxicity(ADCC) mechanisms, both of which require an intact Fc portion of theimmunoglobulin molecule for interaction with effector cell Fc receptorsites on complement proteins. In addition, anti-161P2F10B mAbs thatexert a direct biological effect on tumor growth are useful to treatcancers that express 161P2F10B. Mechanisms by which directly cytotoxicmAbs act include: inhibition of cell growth, modulation of cellulardifferentiation, modulation of tumor angiogenesis factor profiles, andthe induction of apoptosis. The mechanism(s) by which a particularanti-161P2F10B mAb exerts an anti-tumor effect is evaluated using anynumber of in vitro assays that evaluate cell death such as ADCC, ADMMC,complement-mediated cell lysis, and so forth, as is generally known inthe art.

In some patients, the use of murine or other non-human monoclonalantibodies, or human/mouse chimeric mAbs can induce moderate to strongimmune responses against the non-human antibody. This can result inclearance of the antibody from circulation and reduced efficacy. In themost severe cases, such an immune response can lead to the extensiveformation of immune complexes which, potentially, can cause renalfailure. Accordingly, preferred monoclonal antibodies used in thetherapeutic methods of the invention are those that are either fullyhuman or humanized and that bind specifically to the target 161P2F10Bantigen with high affinity but exhibit low or no antigenicity in thepatient.

Therapeutic methods of the invention contemplate the administration ofsingle anti-161P2F10B mAbs as well as combinations, or cocktails, ofdifferent mAbs. Such mAb cocktails can have certain advantages inasmuchas they contain mAbs that target different epitopes, exploit differenteffector mechanisms or combine directly cytotoxic mAbs with mAbs thatrely on immune effector functionality. Such mAbs in combination canexhibit synergistic therapeutic effects. In addition, anti-161P2F10BmAbs can be administered concomitantly with other therapeuticmodalities, including but not limited to various chemotherapeuticagents, androgen-blockers, immune modulators (e.g., IL-2, GM-CSF),surgery or radiation. The anti-161P2F10B mAbs are administered in their“naked” or unconjugated form, or can have a therapeutic agent(s)conjugated to them.

Anti-161P2F10B antibody formulations are administered via any routecapable of delivering the antibodies to a tumor cell. Routes ofadministration include, but are not limited to, intravenous,intraperitoneal, intramuscular, intratumor, intradermal, and the like.Treatment generally involves repeated administration of theanti-161P2F10B antibody preparation, via an acceptable route ofadministration such as intravenous injection (IV), typically at a dosein the range of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg/kg body weight. In general,doses in the range of 10-1000 mg mAb per week are effective and welltolerated.

Based on clinical experience with the Herceptin™ mAb in the treatment ofmetastatic breast cancer, an initial loading dose of approximately 4mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kgIV of the anti-161P2F10B mAb preparation represents an acceptable dosingregimen. Preferably, the initial loading dose is administered as a90-minute or longer infusion. The periodic maintenance dose isadministered as a 30 minute or longer infusion, provided the initialdose was well tolerated. As appreciated by those of skill in the art,various factors can influence the ideal dose regimen in a particularcase. Such factors include, for example, the binding affinity and halflife of the Ab or mAbs used, the degree of 161P2F10B expression in thepatient, the extent of circulating shed 161P2F10B antigen, the desiredsteady-state antibody concentration level, frequency of treatment, andthe influence of chemotherapeutic or other agents used in combinationwith the treatment method of the invention, as well as the health statusof a particular patient.

Optionally, patients should be evaluated for the levels of 161P2F10B ina given sample (e.g. the levels of circulating 161P2F10B antigen and/or161P2F10B expressing cells) in order to assist in the determination ofthe most effective dosing regimen, etc. Such evaluations are also usedfor monitoring purposes throughout therapy, and are useful to gaugetherapeutic success in combination with the evaluation of otherparameters (for example, urine cytology and/or ImmunoCyt levels inbladder cancer therapy, or by analogy, serum PSA levels in prostatecancer therapy).

Anti-idiotypic anti-161P2F10B antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressinga 161P2F10B-related protein. In particular, the generation ofanti-idiotypic antibodies is well known in the art; this methodology canreadily be adapted to generate anti-idiotypic anti-161P2F10B antibodiesthat mimic an epitope on a 161P2F10B-related protein (see, for example,Wagner et al., 1997, Hybridoma 16: 33-40; Foon et al., 1995, J. Clin.Invest. 96:334-342; Herlyn et al., 1996, Cancer Immunol. Immunother.43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccinestrategies.

X.C.) 161P2F10B as a Target for Cellular Immune Responses

Vaccines and methods of preparing vaccines that contain animmunogenically effective amount of one or more HLA-binding peptides asdescribed herein are further embodiments of the invention. Furthermore,vaccines in accordance with the invention encompass compositions of oneor more of the claimed peptides. A peptide can be present in a vaccineindividually. Alternatively, the peptide can exist as a homopolymercomprising multiple copies of the same peptide, or as a heteropolymer ofvarious peptides. Polymers have the advantage of increased immunologicalreaction and, where different peptide epitopes are used to make up thepolymer, the additional ability to induce antibodies and/or CTLs thatreact with different antigenic determinants of the pathogenic organismor tumor-related peptide targeted for an immune response. Thecomposition can be a naturally occurring region of an antigen or can beprepared, e.g., recombinantly or by chemical synthesis.

Carriers that can be used with vaccines of the invention are well knownin the art, and include, e.g., thyroglobulin, albumins such as humanserum albumin, tetanus toxoid, polyamino acids such as poly 1-lysine,poly 1-glutamic acid, influenza, hepatitis B virus core protein, and thelike. The vaccines can contain a physiologically tolerable (i.e.,acceptable) diluent such as water, or saline, preferably phosphatebuffered saline. The vaccines also typically include an adjuvant.Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate,aluminum hydroxide, or alum are examples of materials well known in theart. Additionally, as disclosed herein, CTL responses can be primed byconjugating peptides of the invention to lipids, such astripalmitoyl-S-glycerylcysteinlyseryl-serine (P3 CSS). Moreover, anadjuvant such as a syntheticcytosine-phosphorothiolated-guanine-containing (CpG) oligonucleotideshas been found to increase CTL responses 10- to 100-fold. (see, e.g.Davila and Celis, J. Immunol. 165:539-547 (2000))

Upon immunization with a peptide composition in accordance with theinvention, via injection, aerosol, oral, transdermal, transmucosal,intrapleural, intrathecal, or other suitable routes, the immune systemof the host responds to the vaccine by producing large amounts of CTLsand/or HTLs specific for the desired antigen. Consequently, the hostbecomes at least partially immune to later development of cells thatexpress or overexpress 161P2F10B antigen, or derives at least sometherapeutic benefit when the antigen was tumor-associated.

In some embodiments, it may be desirable to combine the class I peptidecomponents with components that induce or facilitate neutralizingantibody and or helper T cell responses directed to the target antigen.A preferred embodiment of such a composition comprises class I and classII epitopes in accordance with the invention. An alternative embodimentof such a composition comprises a class I and/or class II epitope inaccordance with the invention, along with a cross reactive HTL epitopesuch as PADRE™ (Epimmune, San Diego, Calif.) molecule (described e.g.,in U.S. Pat. No. 5,736,142).

A vaccine of the invention can also include antigen-presenting cells(APC), such as dendritic cells (DC), as a vehicle to present peptides ofthe invention. Vaccine compositions can be created in vitro, followingdendritic cell mobilization and harvesting, whereby loading of dendriticcells occurs in vitro. For example, dendritic cells are transfected,e.g., with a minigene in accordance with the invention, or are pulsedwith peptides. The dendritic cell can then be administered to a patientto elicit immune responses in vivo. Vaccine compositions, either DNA- orpeptide-based, can also be administered in vivo in combination withdendritic cell mobilization whereby loading of dendritic cells occurs invivo.

Preferably, the following principles are utilized when selecting anarray of epitopes for inclusion in a polyepitopic composition for use ina vaccine, or for selecting discrete epitopes to be included in avaccine and/or to be encoded by nucleic acids such as a minigene. It ispreferred that each of the following principles be balanced in order tomake the selection. The multiple epitopes to be incorporated in a givenvaccine composition may be, but need not be, contiguous in sequence inthe native antigen from which the epitopes are derived.

1.) Epitopes are selected which, upon administration, mimic immuneresponses that have been observed to be correlated with tumor clearance.For HLA Class I this includes 3-4 epitopes that come from at least onetumor associated antigen (TAA). For HLA Class II a similar rationale isemployed; again 3-4 epitopes are selected from at least one TAA (see,e.g., Rosenberg et al., Science 278:1447-1450). Epitopes from one TAAmay be used in combination with epitopes from one or more additionalTAAs to produce a vaccine that targets tumors with varying expressionpatterns of frequently-expressed TAAs.

2.) Epitopes are selected that have the requisite binding affinityestablished to be correlated with immunogenicity: for HLA Class I anIC50 of 500 nM or less, often 200 nM or less; and for Class II an IC50of 1000 nM or less.

3.) Sufficient supermotif bearing-peptides, or a sufficient array ofallele-specific motif-bearing peptides, are selected to give broadpopulation coverage. For example, it is preferable to have at least 80%population coverage. A Monte Carlo analysis, a statistical evaluationknown in the art, can be employed to assess the breadth, or redundancyof, population coverage.

4.) When selecting epitopes from cancer-related antigens it is oftenuseful to select analogs because the patient may have developedtolerance to the native epitope.

5.) Of particular relevance are epitopes referred to as “nestedepitopes.” Nested epitopes occur where at least two epitopes overlap ina given peptide sequence. A nested peptide sequence can comprise B cell,HLA class I and/or HLA class II epitopes. When providing nestedepitopes, a general objective is to provide the greatest number ofepitopes per sequence. Thus, an aspect is to avoid providing a peptidethat is any longer than the amino terminus of the amino terminal epitopeand the carboxyl terminus of the carboxyl terminal epitope in thepeptide. When providing a multi-epitopic sequence, such as a sequencecomprising nested epitopes, it is generally important to screen thesequence in order to insure that it does not have pathological or otherdeleterious biological properties.

6.) If a polyepitopic protein is created, or when creating a minigene,an objective is to generate the smallest peptide that encompasses theepitopes of interest. This principle is similar, if not the same as thatemployed when selecting a peptide comprising nested epitopes. However,with an artificial polyepitopic peptide, the size minimization objectiveis balanced against the need to integrate any spacer sequences betweenepitopes in the polyepitopic protein. Spacer amino acid residues can,for example, be introduced to avoid junctional epitopes (an epitoperecognized by the immune system, not present in the target antigen, andonly created by the man-made juxtaposition of epitopes), or tofacilitate cleavage between epitopes and thereby enhance epitopepresentation. Junctional epitopes are generally to be avoided becausethe recipient may generate an immune response to that non-nativeepitope. Of particular concern is a junctional epitope that is a“dominant epitope.” A dominant epitope may lead to such a zealousresponse that immune responses to other epitopes are diminished orsuppressed.

7.) Where the sequences of multiple variants of the same target proteinare present, potential peptide epitopes can also be selected on thebasis of their conservancy. For example, a criterion for conservancy maydefine that the entire sequence of an HLA class I binding peptide or theentire 9-mer core of a class II binding peptide be conserved in adesignated percentage of the sequences evaluated for a specific proteinantigen.

X.C.1. Minigene Vaccines

A number of different approaches are available which allow simultaneousdelivery of multiple epitopes. Nucleic acids encoding the peptides ofthe invention are a particularly useful embodiment of the invention.Epitopes for inclusion in a minigene are preferably selected accordingto the guidelines set forth in the previous section. A preferred meansof administering nucleic acids encoding the peptides of the inventionuses minigene constructs encoding a peptide comprising one or multipleepitopes of the invention.

The use of multi-epitope minigenes is described below and in, Ishioka etal., J. Immunol. 162:3915-3925, 1999; An, L. and Whitton, J. L., J.Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822, 1996;Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine16:426, 1998. For example, a multi-epitope DNA plasmid encodingsupermotif- and/or motif-bearing epitopes derived 161P2F10B, the PADRE®universal helper T cell epitope or multiple HTL epitopes from 161P2F10B(see e.g., Tables VIII-XXI and XXII to XLIX), and an endoplasmicreticulum-translocating signal sequence can be engineered. A vaccine mayalso comprise epitopes that are derived from other TAAs.

The immunogenicity of a multi-epitopic minigene can be confirmed intransgenic mice to evaluate the magnitude of CTL induction responsesagainst the epitopes tested. Further, the immunogenicity of DNA-encodedepitopes in vivo can be correlated with the in vitro responses ofspecific CTL lines against target cells transfected with the DNAplasmid. Thus, these experiments can show that the minigene serves toboth: 1.) generate a CTL response and 2.) that the induced CTLsrecognized cells expressing the encoded epitopes.

For example, to create a DNA sequence encoding the selected epitopes(minigene) for expression in human cells, the amino acid sequences ofthe epitopes may be reverse translated. A human codon usage table can beused to guide the codon choice for each amino acid. Theseepitope-encoding DNA sequences may be directly adjoined, so that whentranslated, a continuous polypeptide sequence is created. To optimizeexpression and/or immunogenicity, additional elements can beincorporated into the minigene design. Examples of amino acid sequencesthat can be reverse translated and included in the minigene sequenceinclude: HLA class I epitopes, HLA class II epitopes, antibody epitopes,a ubiquitination signal sequence, and/or an endoplasmic reticulumtargeting signal. In addition, HLA presentation of CTL and HTL epitopesmay be improved by including synthetic (e.g. poly-alanine) ornaturally-occurring flanking sequences adjacent to the CTL or HTLepitopes; these larger peptides comprising the epitope(s) are within thescope of the invention.

The minigene sequence may be converted to DNA by assemblingoligonucleotides that encode the plus and minus strands of the minigene.Overlapping oligonucleotides (30-100 bases long) may be synthesized,phosphorylated, purified and annealed under appropriate conditions usingwell known techniques. The ends of the oligonucleotides can be joined,for example, using T4 DNA ligase. This synthetic minigene, encoding theepitope polypeptide, can then be cloned into a desired expressionvector.

Standard regulatory sequences well known to those of skill in the artare preferably included in the vector to ensure expression in the targetcells. Several vector elements are desirable: a promoter with adown-stream cloning site for minigene insertion; a polyadenylationsignal for efficient transcription termination; an E. coli origin ofreplication; and an E. coli selectable marker (e.g. ampicillin orkanamycin resistance). Numerous promoters can be used for this purpose,e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat.Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

Additional vector modifications may be desired to optimize minigeneexpression and immunogenicity. In some cases, introns are required forefficient gene expression, and one or more synthetic ornaturally-occurring introns could be incorporated into the transcribedregion of the minigene. The inclusion of mRNA stabilization sequencesand sequences for replication in mammalian cells may also be consideredfor increasing minigene expression.

Once an expression vector is selected, the minigene is cloned into thepolylinker region downstream of the promoter. This plasmid istransformed into an appropriate E. coli strain, and DNA is preparedusing standard techniques. The orientation and DNA sequence of theminigene, as well as all other elements included in the vector, areconfirmed using restriction mapping and DNA sequence analysis. Bacterialcells harboring the correct plasmid can be stored as a master cell bankand a working cell bank.

In addition, immunostimulatory sequences (ISSs or CpGs) appear to play arole in the immunogenicity of DNA vaccines. These sequences may beincluded in the vector, outside the minigene coding sequence, if desiredto enhance immunogenicity.

In some embodiments, a bi-cistronic expression vector which allowsproduction of both the minigene-encoded epitopes and a second protein(included to enhance or decrease immunogenicity) can be used. Examplesof proteins or polypeptides that could beneficially enhance the immuneresponse if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF),cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, orfor HTL responses, pan-DR binding proteins (PADRE™, Epimmune, San Diego,Calif.). Helper (HTL) epitopes can be joined to intracellular targetingsignals and expressed separately from expressed CTL epitopes; thisallows direction of the HTL epitopes to a cell compartment differentthan that of the CTL epitopes. If required, this could facilitate moreefficient entry of HTL epitopes into the HLA class II pathway, therebyimproving HTL induction. In contrast to HTL or CTL induction,specifically decreasing the immune response by co-expression ofimmunosuppressive molecules (e.g. TGF-β) may be beneficial in certaindiseases.

Therapeutic quantities of plasmid DNA can be produced for example, byfermentation in E. coli, followed by purification. Aliquots from theworking cell bank are used to inoculate growth medium, and grown tosaturation in shaker flasks or a bioreactor according to well-knowntechniques. Plasmid DNA can be purified using standard bioseparationtechnologies such as solid phase anion-exchange resins supplied byQIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can beisolated from the open circular and linear forms using gelelectrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using a variety offormulations. The simplest of these is reconstitution of lyophilized DNAin sterile phosphate-buffer saline (PBS). This approach, known as “nakedDNA,” is currently being used for intramuscular (IM) administration inclinical trials. To maximize the immunotherapeutic effects of minigeneDNA vaccines, an alternative method for formulating purified plasmid DNAmay be desirable. A variety of methods have been described, and newtechniques may become available. Cationic lipids, glycolipids, andfusogenic liposomes can also be used in the formulation (see, e.g., asdescribed by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7):682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner, et al.,Proc. Nat'l Acad. Sci. USA 84:7413 (1987). In addition, peptides andcompounds referred to collectively as protective, interactive,non-condensing compounds (PINC) could also be complexed to purifiedplasmid DNA to influence variables such as stability, intramusculardispersion, or trafficking to specific organs or cell types.

Target cell sensitization can be used as a functional assay forexpression and HLA class I presentation of minigene-encoded CTLepitopes. For example, the plasmid DNA is introduced into a mammaliancell line that is suitable as a target for standard CTL chromium releaseassays. The transfection method used will be dependent on the finalformulation. Electroporation can be used for “naked” DNA, whereascationic lipids allow direct in vitro transfection. A plasmid expressinggreen fluorescent protein (GFP) can be co-transfected to allowenrichment of transfected cells using fluorescence activated cellsorting (FACS). These cells are then chromium-51 (51 Cr) labeled andused as target cells for epitope-specific CTL lines; cytolysis, detectedby 51 Cr release, indicates both production of, and HLA presentation of,minigene-encoded CTL epitopes. Expression of HTL epitopes may beevaluated in an analogous manner using assays to assess HTL activity.

In vivo immunogenicity is a second approach for functional testing ofminigene DNA formulations. Transgenic mice expressing appropriate humanHLA proteins are immunized with the DNA product. The dose and route ofadministration are formulation dependent (e.g., IM for DNA in PBS,intraperitoneal (i.p.) for lipid-complexed DNA). Twenty-one days afterimmunization, splenocytes are harvested and restimulated for one week inthe presence of peptides encoding each epitope being tested. Thereafter,for CTL effector cells, assays are conducted for cytolysis ofpeptide-loaded, 51 Cr-labeled target cells using standard techniques.Lysis of target cells that were sensitized by HLA loaded with peptideepitopes, corresponding to minigene-encoded epitopes, demonstrates DNAvaccine function for in vivo induction of CTLs Immunogenicity of HTLepitopes is confirmed in transgenic mice in an analogous manner.

Alternatively, the nucleic acids can be administered using ballisticdelivery as described, for instance, in U.S. Pat. No. 5,204,253. Usingthis technique, particles comprised solely of DNA are administered. In afurther alternative embodiment, DNA can be adhered to particles, such asgold particles.

Minigenes can also be delivered using other bacterial or viral deliverysystems well known in the art, e.g., an expression construct encodingepitopes of the invention can be incorporated into a viral vector suchas vaccinia.

X.C.2. Combinations of CTL Peptides with Helper Peptides

Vaccine compositions comprising CTL peptides of the invention can bemodified, e.g., analoged, to provide desired attributes, such asimproved serum half life, broadened population coverage or enhancedimmunogenicity.

For instance, the ability of a peptide to induce CTL activity can beenhanced by linking the peptide to a sequence which contains at leastone epitope that is capable of inducing a T helper cell response.Although a CTL peptide can be directly linked to a T helper peptide,often CTL epitope/HTL epitope conjugates are linked by a spacermolecule. The spacer is typically comprised of relatively small, neutralmolecules, such as amino acids or amino acid mimetics, which aresubstantially uncharged under physiological conditions. The spacers aretypically selected from, e.g., Ala, Gly, or other neutral spacers ofnonpolar amino acids or neutral polar amino acids. It will be understoodthat the optionally present spacer need not be comprised of the sameresidues and thus may be a hetero- or homo-oligomer. When present, thespacer will usually be at least one or two residues, more usually threeto six residues and sometimes 10 or more residues. The CTL peptideepitope can be linked to the T helper peptide epitope either directly orvia a spacer either at the amino or carboxy terminus of the CTL peptide.The amino terminus of either the immunogenic peptide or the T helperpeptide may be acylated.

In certain embodiments, the T helper peptide is one that is recognizedby T helper cells present in a majority of a genetically diversepopulation. This can be accomplished by selecting peptides that bind tomany, most, or all of the HLA class II molecules. Examples of such aminoacid bind many HLA Class II molecules include sequences from antigenssuch as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO:25), Plasmodium falciparum circumsporozoite (CS) protein at positions378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 26), and Streptococcus 18 kDprotein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO: 27). Otherexamples include peptides bearing a DR 1-4-7 supermotif, or either ofthe DR3 motifs.

Alternatively, it is possible to prepare synthetic peptides capable ofstimulating T helper lymphocytes, in a loosely HLA-restricted fashion,using amino acid sequences not found in nature (see, e.g., PCTpublication WO 95/07707). These synthetic compounds calledPan-DR-binding epitopes (e.g., PADRE™, Epimmune, Inc., San Diego,Calif.) are designed, most preferably, to bind most HLA-DR (human HLAclass II) molecules. For instance, a pan-DR-binding epitope peptidehaving the formula: aKXVAAWTLKAAa (SEQ ID NO: 28), where “X” is eithercyclohexylalanine, phenylalanine, or tyrosine, and a is either d-alanineor 1-alanine, has been found to bind to most HLA-DR alleles, and tostimulate the response of T helper lymphocytes from most individuals,regardless of their HLA type. An alternative of a pan-DR binding epitopecomprises all “L” natural amino acids and can be provided in the form ofnucleic acids that encode the epitope.

HTL peptide epitopes can also be modified to alter their biologicalproperties. For example, they can be modified to include d-amino acidsto increase their resistance to proteases and thus extend their serumhalf life, or they can be conjugated to other molecules such as lipids,proteins, carbohydrates, and the like to increase their biologicalactivity. For example, a T helper peptide can be conjugated to one ormore palmitic acid chains at either the amino or carboxyl termini

X.C.3. Combinations of CTL Peptides with T Cell Priming Agents

In some embodiments it may be desirable to include in the pharmaceuticalcompositions of the invention at least one component which primes Blymphocytes or T lymphocytes. Lipids have been identified as agentscapable of priming CTL in vivo. For example, palmitic acid residues canbe attached to the ε- and α-amino groups of a lysine residue and thenlinked, e.g., via one or more linking residues such as Gly, Gly-Gly-,Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidatedpeptide can then be administered either directly in a micelle orparticle, incorporated into a liposome, or emulsified in an adjuvant,e.g., incomplete Freund's adjuvant. In a preferred embodiment, aparticularly effective immunogenic composition comprises palmitic acidattached to ε- and α-amino groups of Lys, which is attached via linkage,e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.

As another example of lipid priming of CTL responses, E. colilipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine(P₃CSS) can be used to prime virus specific CTL when covalently attachedto an appropriate peptide (see, e.g., Deres, et al., Nature 342:561,1989). Peptides of the invention can be coupled to P₃CSS, for example,and the lipopeptide administered to an individual to prime specificallyan immune response to the target antigen. Moreover, because theinduction of neutralizing antibodies can also be primed withP₃CSS-conjugated epitopes, two such compositions can be combined to moreeffectively elicit both humoral and cell-mediated responses.

X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTLPeptides

An embodiment of a vaccine composition in accordance with the inventioncomprises ex vivo administration of a cocktail of epitope-bearingpeptides to PBMC, or isolated DC therefrom, from the patient's blood. Apharmaceutical to facilitate harvesting of DC can be used, such asProgenipoietin™ (Pharmacia-Monsanto, St. Louis, Mo.) or GM-CSF/IL-4.After pulsing the DC with peptides and prior to reinfusion intopatients, the DC are washed to remove unbound peptides. In thisembodiment, a vaccine comprises peptide-pulsed DCs which present thepulsed peptide epitopes complexed with HLA molecules on their surfaces.

The DC can be pulsed ex vivo with a cocktail of peptides, some of whichstimulate CTL responses to 161P2F10B. Optionally, a helper T cell (HTL)peptide, such as a natural or artificial loosely restricted HLA Class IIpeptide, can be included to facilitate the CTL response. Thus, a vaccinein accordance with the invention is used to treat a cancer whichexpresses or overexpresses 161P2F10B.

X.D. Adoptive Immunotherapy

Antigenic 161P2F10B-related peptides are used to elicit a CTL and/or HTLresponse ex vivo, as well. The resulting CTL or HTL cells, can be usedto treat tumors in patients that do not respond to other conventionalforms of therapy, or will not respond to a therapeutic vaccine peptideor nucleic acid in accordance with the invention. Ex vivo CTL or HTLresponses to a particular antigen are induced by incubating in tissueculture the patient's, or genetically compatible, CTL or HTL precursorcells together with a source of antigen-presenting cells (APC), such asdendritic cells, and the appropriate immunogenic peptide. After anappropriate incubation time (typically about 7-28 days), in which theprecursor cells are activated and expanded into effector cells, thecells are infused back into the patient, where they will destroy (CTL)or facilitate destruction (HTL) of their specific target cell (e.g., atumor cell). Transfected dendritic cells may also be used as antigenpresenting cells.

X.E. Administration of Vaccines for Therapeutic or Prophylactic Purposes

Pharmaceutical and vaccine compositions of the invention are typicallyused to treat and/or prevent a cancer that expresses or overexpresses161P2F10B. In therapeutic applications, peptide and/or nucleic acidcompositions are administered to a patient in an amount sufficient toelicit an effective B cell, CTL and/or HTL response to the antigen andto cure or at least partially arrest or slow symptoms and/orcomplications. An amount adequate to accomplish this is defined as“therapeutically effective dose.” Amounts effective for this use willdepend on, e.g., the particular composition administered, the manner ofadministration, the stage and severity of the disease being treated, theweight and general state of health of the patient, and the judgment ofthe prescribing physician.

For pharmaceutical compositions, the immunogenic peptides of theinvention, or DNA encoding them, are generally administered to anindividual already bearing a tumor that expresses 161P2F10B. Thepeptides or DNA encoding them can be administered individually or asfusions of one or more peptide sequences. Patients can be treated withthe immunogenic peptides separately or in conjunction with othertreatments, such as surgery, as appropriate.

For therapeutic use, administration should generally begin at the firstdiagnosis of 161P2F10B-associated cancer. This is followed by boostingdoses until at least symptoms are substantially abated and for a periodthereafter. The embodiment of the vaccine composition (i.e., including,but not limited to embodiments such as peptide cocktails, polyepitopicpolypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic cells)delivered to the patient may vary according to the stage of the diseaseor the patient's health status. For example, in a patient with a tumorthat expresses 161P2F10B, a vaccine comprising 161P2F10B-specific CTLmay be more efficacious in killing tumor cells in patient with advanceddisease than alternative embodiments.

It is generally important to provide an amount of the peptide epitopedelivered by a mode of administration sufficient to stimulateeffectively a cytotoxic T cell response; compositions which stimulatehelper T cell responses can also be given in accordance with thisembodiment of the invention.

The dosage for an initial therapeutic immunization generally occurs in aunit dosage range where the lower value is about 1, 5, 50, 500, or 1,000μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg.Dosage values for a human typically range from about 500 μg to about50,000 μg per 70 kilogram patient. Boosting dosages of between about 1.0μg to about 50,000 μg of peptide pursuant to a boosting regimen overweeks to months may be administered depending upon the patient'sresponse and condition as determined by measuring the specific activityof CTL and HTL obtained from the patient's blood. Administration shouldcontinue until at least clinical symptoms or laboratory tests indicatethat the neoplasia, has been eliminated or reduced and for a periodthereafter. The dosages, routes of administration, and dose schedulesare adjusted in accordance with methodologies known in the art.

In certain embodiments, the peptides and compositions of the presentinvention are employed in serious disease states, that is,life-threatening or potentially life threatening situations. In suchcases, as a result of the minimal amounts of extraneous substances andthe relative nontoxic nature of the peptides in preferred compositionsof the invention, it is possible and may be felt desirable by thetreating physician to administer substantial excesses of these peptidecompositions relative to these stated dosage amounts.

The vaccine compositions of the invention can also be used purely asprophylactic agents. Generally the dosage for an initial prophylacticimmunization generally occurs in a unit dosage range where the lowervalue is about 1, 5, 50, 500, or 1000 μg and the higher value is about10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a humantypically range from about 500 μg to about 50,000 μg per 70 kilogrampatient. This is followed by boosting dosages of between about 1.0 μg toabout 50,000 μg of peptide administered at defined intervals from aboutfour weeks to six months after the initial administration of vaccine.The immunogenicity of the vaccine can be assessed by measuring thespecific activity of CTL and HTL obtained from a sample of the patient'sblood.

The pharmaceutical compositions for therapeutic treatment are intendedfor parenteral, topical, oral, nasal, intrathecal, or local (e.g. as acream or topical ointment) administration. Preferably, thepharmaceutical compositions are administered parentally, e.g.,intravenously, subcutaneously, intradermally, or intramuscularly. Thus,the invention provides compositions for parenteral administration whichcomprise a solution of the immunogenic peptides dissolved or suspendedin an acceptable carrier, preferably an aqueous carrier.

A variety of aqueous carriers may be used, e.g., water, buffered water,0.8% saline, 0.3% glycine, hyaluronic acid and the like. Thesecompositions may be sterilized by conventional, well-known sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration.

The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH-adjusting and buffering agents, tonicity adjusting agents, wettingagents, preservatives, and the like, for example, sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride, sorbitanmonolaurate, triethanolamine oleate, etc.

The concentration of peptides of the invention in the pharmaceuticalformulations can vary widely, i.e., from less than about 0.1%, usuallyat or at least about 2% to as much as 20% to 50% or more by weight, andwill be selected primarily by fluid volumes, viscosities, etc., inaccordance with the particular mode of administration selected.

A human unit dose form of a composition is typically included in apharmaceutical composition that comprises a human unit dose of anacceptable carrier, in one embodiment an aqueous carrier, and isadministered in a volume/quantity that is known by those of skill in theart to be used for administration of such compositions to humans (see,e.g., Remington's Pharmaceutical Sciences, 17th Edition, A. Gennaro,Editor, Mack Publishing Co., Easton, Pa., 1985). For example a peptidedose for initial immunization can be from about 1 to about 50,000 μg,generally 100-5,000 μg, for a 70 kg patient. For example, for nucleicacids an initial immunization may be performed using an expressionvector in the form of naked nucleic acid administered IM (or SC or ID)in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to1000 μg) can also be administered using a gene gun. Following anincubation period of 3-4 weeks, a booster dose is then administered. Thebooster can be recombinant fowlpox virus administered at a dose of 5-107to 5×109 pfu.

For antibodies, a treatment generally involves repeated administrationof the anti-161P2F10B antibody preparation, via an acceptable route ofadministration such as intravenous injection (IV), typically at a dosein the range of about 0.1 to about 10 mg/kg body weight. In general,doses in the range of 10-500 mg mAb per week are effective and welltolerated. Moreover, an initial loading dose of approximately 4 mg/kgpatient body weight IV, followed by weekly doses of about 2 mg/kg IV ofthe anti-161P2F10B mAb preparation represents an acceptable dosingregimen. As appreciated by those of skill in the art, various factorscan influence the ideal dose in a particular case. Such factors include,for example, half life of a composition, the binding affinity of an Ab,the immunogenicity of a substance, the degree of 161P2F10B expression inthe patient, the extent of circulating shed 161P2F10B antigen, thedesired steady-state concentration level, frequency of treatment, andthe influence of chemotherapeutic or other agents used in combinationwith the treatment method of the invention, as well as the health statusof a particular patient. Non-limiting preferred human unit doses are,for example, 500 μg-1 mg, 1 mg-50 mg, 50 mg-100 mg, 100 mg-200 mg, 200mg-300 mg, 400 mg-500 mg, 500 mg-600 mg, 600 mg-700 mg, 700 mg -800 mg,800 mg-900 mg, 900 mg-1 g, or 1 mg-700 mg. In certain embodiments, thedose is in a range of 2-5 mg/kg body weight, e.g., with follow on weeklydoses of 1-3 mg/kg; 0.5 mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/kg bodyweight followed, e.g., in two, three or four weeks by weekly doses;0.5-10 mg/kg body weight, e.g., followed in two, three or four weeks byweekly doses; 225, 250, 275, 300, 325, 350, 375, 400 mg m2 of body areaweekly; 1-600 mg m2 of body area weekly; 225-400 mg m2 of body areaweekly; these does can be followed by weekly doses for 2, 3, 4, 5, 6, 7,8, 9, 19, 11, 12 or more weeks.

In one embodiment, human unit dose forms of polynucleotides comprise asuitable dosage range or effective amount that provides any therapeuticeffect. As appreciated by one of ordinary skill in the art a therapeuticeffect depends on a number of factors, including the sequence of thepolynucleotide, molecular weight of the polynucleotide and route ofadministration. Dosages are generally selected by the physician or otherhealth care professional in accordance with a variety of parametersknown in the art, such as severity of symptoms, history of the patientand the like. Generally, for a polynucleotide of about 20 bases, adosage range may be selected from, for example, an independentlyselected lower limit such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 mg/kg up to anindependently selected upper limit, greater than the lower limit, ofabout 60, 80, 100, 200, 300, 400, 500, 750, 1000, 1500, 2000, 3000,4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For example, a dosemay be about any of the following: 0.1 to 100 mg/kg, 0.1 to 50 mg/kg,0.1 to 25 mg/kg, 0.1 to 10 mg/kg, 1 to 500 mg/kg, 100 to 400 mg/kg, 200to 300 mg/kg, 1 to 100 mg/kg, 100 to 200 mg/kg, 300 to 400 mg/kg, 400 to500 mg/kg, 500 to 1000 mg/kg, 500 to 5000 mg/kg, or 500 to 10,000 mg/kg.Generally, parenteral routes of administration may require higher dosesof polynucleotide compared to more direct application to the nucleotideto diseased tissue, as do polynucleotides of increasing length.

In one embodiment, human unit dose forms of T-cells comprise a suitabledosage range or effective amount that provides any therapeutic effect.As appreciated by one of ordinary skill in the art, a therapeutic effectdepends on a number of factors. Dosages are generally selected by thephysician or other health care professional in accordance with a varietyof parameters known in the art, such as severity of symptoms, history ofthe patient and the like. A dose may be about 104 cells to about 106cells, about 106 cells to about 108 cells, about 108 to about 1011cells, or about 108 to about 5×1010 cells. A dose may also about 106cells/m2 to about 1010 cells/m2, or about 106 cells/m2 to about 108cells/m2.

Proteins(s) of the invention, and/or nucleic acids encoding theprotein(s), can also be administered via liposomes, which may also serveto: 1) target the proteins(s) to a particular tissue, such as lymphoidtissue; 2) to target selectively to diseases cells; or, 3) to increasethe half-life of the peptide composition. Liposomes include emulsions,foams, micelles, insoluble monolayers, liquid crystals, phospholipiddispersions, lamellar layers and the like. In these preparations, thepeptide to be delivered is incorporated as part of a liposome, alone orin conjunction with a molecule which binds to a receptor prevalent amonglymphoid cells, such as monoclonal antibodies which bind to the CD45antigen, or with other therapeutic or immunogenic compositions. Thus,liposomes either filled or decorated with a desired peptide of theinvention can be directed to the site of lymphoid cells, where theliposomes then deliver the peptide compositions. Liposomes for use inaccordance with the invention are formed from standard vesicle-forminglipids, which generally include neutral and negatively chargedphospholipids and a sterol, such as cholesterol. The selection of lipidsis generally guided by consideration of, e.g., liposome size, acidlability and stability of the liposomes in the blood stream. A varietyof methods are available for preparing liposomes, as described in, e.g.,Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Pat.Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

For targeting cells of the immune system, a ligand to be incorporatedinto the liposome can include, e.g., antibodies or fragments thereofspecific for cell surface determinants of the desired immune systemcells. A liposome suspension containing a peptide may be administeredintravenously, locally, topically, etc. in a dose which varies accordingto, inter alia, the manner of administration, the peptide beingdelivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, one or more peptides of the invention, and morepreferably at a concentration of 25%-75%.

For aerosol administration, immunogenic peptides are preferably suppliedin finely divided form along with a surfactant and propellant. Typicalpercentages of peptides are about 0.01%-20% by weight, preferably about1%-10%. The surfactant must, of course, be nontoxic, and preferablysoluble in the propellant. Representative of such agents are the estersor partial esters of fatty acids containing from about 6 to 22 carbonatoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic,linolenic, olesteric and oleic acids with an aliphatic polyhydricalcohol or its cyclic anhydride. Mixed esters, such as mixed or naturalglycerides may be employed. The surfactant may constitute about 0.1%-20%by weight of the composition, preferably about 0.25-5%. The balance ofthe composition is ordinarily propellant. A carrier can also beincluded, as desired, as with, e.g., lecithin for intranasal delivery.

XI.) Diagnostic and Prognostic Embodiments of 161P2F10B

As disclosed herein, 161P2F10B polynucleotides, polypeptides, reactivecytotoxic T cells (CTL), reactive helper T cells (HTL) andanti-polypeptide antibodies are used in well known diagnostic,prognostic and therapeutic assays that examine conditions associatedwith dysregulated cell growth such as cancer, in particular the cancerslisted in Table I (see, e.g., both its specific pattern of tissueexpression as well as its overexpression in certain cancers as describedfor example in the Example entitled “Expression analysis of 161P2F10B innormal tissues, and patient specimens”).

161P2F10B can be analogized to a prostate associated antigen PSA, thearchetypal marker that has been used by medical practitioners for yearsto identify and monitor the presence of prostate cancer (see, e.g.,Merrill et al., J. Urol. 163(2): 503-5120 (2000); Polascik et al., J.Urol. August; 162(2):293-306 (1999) and Fortier et al., J. Nat. CancerInst. 91(19): 1635-1640(1999)). A variety of other diagnostic markersare also used in similar contexts including p53 and K-ras (see, e.g.,Tulchinsky et al., Int J Mol Med 1999 Jul. 4(1):99-102 and Minimoto etal., Cancer Detect Prey 2000; 24(1):1-12). Therefore, this disclosure of161P2F10B polynucleotides and polypeptides (as well as 161P2F10Bpolynucleotide probes and anti-161P2F10B antibodies used to identify thepresence of these molecules) and their properties allows skilledartisans to utilize these molecules in methods that are analogous tothose used, for example, in a variety of diagnostic assays directed toexamining conditions associated with cancer.

Typical embodiments of diagnostic methods which utilize the 161P2F10Bpolynucleotides, polypeptides, reactive T cells and antibodies areanalogous to those methods from well-established diagnostic assays,which employ, e.g., PSA polynucleotides, polypeptides, reactive T cellsand antibodies. For example, just as PSA polynucleotides are used asprobes (for example in Northern analysis, see, e.g., Sharief et al.,Biochem. Mol. Biol. Int. 33(3):567-74(1994)) and primers (for example inPCR analysis, see, e.g., Okegawa et al., J. Urol. 163(4): 1189-1190(2000)) to observe the presence and/or the level of PSA mRNAs in methodsof monitoring PSA overexpression or the metastasis of prostate cancers,the 161P2F10B polynucleotides described herein can be utilized in thesame way to detect 161P2F10B overexpression or the metastasis ofprostate and other cancers expressing this gene. Alternatively, just asPSA polypeptides are used to generate antibodies specific for PSA whichcan then be used to observe the presence and/or the level of PSAproteins in methods to monitor PSA protein overexpression (see, e.g.,Stephan et al., Urology 55(4):560-3 (2000)) or the metastasis ofprostate cells (see, e.g., Alanen et al., Pathol. Res. Pract.192(3):233-7 (1996)), the 161P2F10B polypeptides described herein can beutilized to generate antibodies for use in detecting 161P2F10Boverexpression or the metastasis of prostate cells and cells of othercancers expressing this gene.

Specifically, because metastases involves the movement of cancer cellsfrom an organ of origin (such as the lung or prostate gland etc.) to adifferent area of the body (such as a lymph node), assays which examinea biological sample for the presence of cells expressing 161P2F10Bpolynucleotides and/or polypeptides can be used to provide evidence ofmetastasis. For example, when a biological sample from tissue that doesnot normally contain 161P2F10B-expressing cells (lymph node) is found tocontain 161P2F10B-expressing cells such as the 161P2F10B expression seenin LAPC4 and LAPC9, xenografts isolated from lymph node and bonemetastasis, respectively, this finding is indicative of metastasis.

Alternatively 161P2F10B polynucleotides and/or polypeptides can be usedto provide evidence of cancer, for example, when cells in a biologicalsample that do not normally express 161P2F10B or express 161P2F10B at adifferent level are found to express 161P2F10B or have an increasedexpression of 161P2F10B (see, e.g., the 161P2F10B expression in thecancers listed in Table I and in patient samples etc. shown in theaccompanying Figures). In such assays, artisans may further wish togenerate supplementary evidence of metastasis by testing the biologicalsample for the presence of a second tissue restricted marker (inaddition to 161P2F10B) such as PSA, PSCA etc. (see, e.g., Alanen et al.,Pathol. Res. Pract. 192(3): 233-237 (1996)).

Just as PSA polynucleotide fragments and polynucleotide variants areemployed by skilled artisans for use in methods of monitoring PSA,161P2F10B polynucleotide fragments and polynucleotide variants are usedin an analogous manner. In particular, typical PSA polynucleotides usedin methods of monitoring PSA are probes or primers which consist offragments of the PSA cDNA sequence. Illustrating this, primers used toPCR amplify a PSA polynucleotide must include less than the whole PSAsequence to function in the polymerase chain reaction. In the context ofsuch PCR reactions, skilled artisans generally create a variety ofdifferent polynucleotide fragments that can be used as primers in orderto amplify different portions of a polynucleotide of interest or tooptimize amplification reactions (see, e.g., Caetano-Anolles, G.Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al., MethodsMol. Biol. 98:121-154 (1998)). An additional illustration of the use ofsuch fragments is provided in the Example entitled “Expression analysisof 161P2F10B in normal tissues, and patient specimens,” where a161P2F10B polynucleotide fragment is used as a probe to show theexpression of 161P2F10B RNAs in cancer cells. In addition, variantpolynucleotide sequences are typically used as primers and probes forthe corresponding mRNAs in PCR and Northern analyses (see, e.g., Sawaiet al., Fetal Diagn. Ther. 1996 November-December 11(6):407-13 andCurrent Protocols In Molecular Biology, Volume 2, Unit 2, Frederick M.Ausubel et al. eds., 1995)). Polynucleotide fragments and variants areuseful in this context where they are capable of binding to a targetpolynucleotide sequence (e.g., a 161P2F10B polynucleotide shown in FIG.2 or variant thereof) under conditions of high stringency.

Furthermore, PSA polypeptides which contain an epitope that can berecognized by an antibody or T cell that specifically binds to thatepitope are used in methods of monitoring PSA. 161P2F10B polypeptidefragments and polypeptide analogs or variants can also be used in ananalogous manner. This practice of using polypeptide fragments orpolypeptide variants to generate antibodies (such as anti-PSA antibodiesor T cells) is typical in the art with a wide variety of systems such asfusion proteins being used by practitioners (see, e.g., CurrentProtocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubelet al. eds., 1995). In this context, each epitope(s) functions toprovide the architecture with which an antibody or T cell is reactive.Typically, skilled artisans create a variety of different polypeptidefragments that can be used in order to generate immune responsesspecific for different portions of a polypeptide of interest (see, e.g.,U.S. Pat. Nos. 5,840,501 and 5,939,533). For example it may bepreferable to utilize a polypeptide comprising one of the 161P2F10Bbiological motifs discussed herein or a motif-bearing subsequence whichis readily identified by one of skill in the art based on motifsavailable in the art. Polypeptide fragments, variants or analogs aretypically useful in this context as long as they comprise an epitopecapable of generating an antibody or T cell specific for a targetpolypeptide sequence (e.g. a 161P2F10B polypeptide shown in FIG. 3).

As shown herein, the 161P2F10B polynucleotides and polypeptides (as wellas the 161P2F10B polynucleotide probes and anti-161P2F10B antibodies orT cells used to identify the presence of these molecules) exhibitspecific properties that make them useful in diagnosing cancers such asthose listed in Table I. Diagnostic assays that measure the presence of161P2F10B gene products, in order to evaluate the presence or onset of adisease condition described herein, such as prostate cancer, are used toidentify patients for preventive measures or further monitoring, as hasbeen done so successfully with PSA. Moreover, these materials satisfy aneed in the art for molecules having similar or complementarycharacteristics to PSA in situations where, for example, a definitediagnosis of metastasis of prostatic origin cannot be made on the basisof a test for PSA alone (see, e.g., Alanen et al., Pathol. Res. Pract.192(3): 233-237 (1996)), and consequently, materials such as 161P2F10Bpolynucleotides and polypeptides (as well as the 161P2F10Bpolynucleotide probes and anti-161P2F10B antibodies used to identify thepresence of these molecules) need to be employed to confirm a metastasesof prostatic origin.

Finally, in addition to their use in diagnostic assays, the 161P2F10Bpolynucleotides disclosed herein have a number of other utilities suchas their use in the identification of oncogenetic associated chromosomalabnormalities in the chromosomal region to which the 161P2F10B gene maps(see the Example entitled “Chromosomal Mapping of 161P2F10B” below).Moreover, in addition to their use in diagnostic assays, the161P2F10B-related proteins and polynucleotides disclosed herein haveother utilities such as their use in the forensic analysis of tissues ofunknown origin (see, e.g., Takahama K Forensic Sci Int 1996 Jun. 28;80(1-2): 63-9).

Additionally, 161P2F10B-related proteins or polynucleotides of theinvention can be used to treat a pathologic condition characterized bythe over-expression of 161P2F10B. For example, the amino acid or nucleicacid sequence of FIG. 2 or FIG. 3, or fragments of either, can be usedto generate an immune response to a 161P2F10B antigen. Antibodies orother molecules that react with 161P2F10B can be used to modulate thefunction of this molecule, and thereby provide a therapeutic benefit.

XII.) Inhibition of 161P2F10B Protein Function

The invention includes various methods and compositions for inhibitingthe binding of 161P2F10B to its binding partner or its association withother protein(s) as well as methods for inhibiting 161P2F10B function.

XII.A.) Inhibition of 161P2F10B With Intracellular Antibodies

In one approach, a recombinant vector that encodes single chainantibodies that specifically bind to 161P2F10B are introduced into161P2F10B expressing cells via gene transfer technologies. Accordingly,the encoded single chain anti-161P2F10B antibody is expressedintracellularly, binds to 161P2F10B protein, and thereby inhibits itsfunction. Methods for engineering such intracellular single chainantibodies are well known. Such intracellular antibodies, also known as“intrabodies”, are specifically targeted to a particular compartmentwithin the cell, providing control over where the inhibitory activity ofthe treatment is focused. This technology has been successfully appliedin the art (for review, see Richardson and Marasco, 1995, TIBTECH vol.13). Intrabodies have been shown to virtually eliminate the expressionof otherwise abundant cell surface receptors (see, e.g., Richardson etal., 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al.,1994, J. Biol. Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther.1: 332-337).

Single chain antibodies comprise the variable domains of the heavy andlight chain joined by a flexible linker polypeptide, and are expressedas a single polypeptide. Optionally, single chain antibodies areexpressed as a single chain variable region fragment joined to the lightchain constant region. Well-known intracellular trafficking signals areengineered into recombinant polynucleotide vectors encoding such singlechain antibodies in order to target precisely the intrabody to thedesired intracellular compartment. For example, intrabodies targeted tothe endoplasmic reticulum (ER) are engineered to incorporate a leaderpeptide and, optionally, a C-terminal ER retention signal, such as theKDEL amino acid motif. Intrabodies intended to exert activity in thenucleus are engineered to include a nuclear localization signal. Lipidmoieties are joined to intrabodies in order to tether the intrabody tothe cytosolic side of the plasma membrane. Intrabodies can also betargeted to exert function in the cytosol. For example, cytosolicintrabodies are used to sequester factors within the cytosol, therebypreventing them from being transported to their natural cellulardestination.

In one embodiment, intrabodies are used to capture 161P2F10B in thenucleus, thereby preventing its activity within the nucleus. Nucleartargeting signals are engineered into such 161P2F10B intrabodies inorder to achieve the desired targeting. Such 161P2F10B intrabodies aredesigned to bind specifically to a particular 161P2F10B domain. Inanother embodiment, cytosolic intrabodies that specifically bind to a161P2F10B protein are used to prevent 161P2F10B from gaining access tothe nucleus, thereby preventing it from exerting any biological activitywithin the nucleus (e.g., preventing 161P2F10B from formingtranscription complexes with other factors).

In order to specifically direct the expression of such intrabodies toparticular cells, the transcription of the intrabody is placed under theregulatory control of an appropriate tumor-specific promoter and/orenhancer. In order to target intrabody expression specifically toprostate, for example, the PSA promoter and/or promoter/enhancer can beutilized (See, for example, U.S. Pat. No. 5,919,652 issued 6 Jul. 1999).

XII.B.) Inhibition of 161P2F10B with Recombinant Proteins

In another approach, recombinant molecules bind to 161P2F10B and therebyinhibit 161P2F10B function. For example, these recombinant moleculesprevent or inhibit 161P2F10B from accessing/binding to its bindingpartner(s) or associating with other protein(s). Such recombinantmolecules can, for example, contain the reactive part(s) of a 161P2F10Bspecific antibody molecule. In a particular embodiment, the 161P2F10Bbinding domain of a 161P2F10B binding partner is engineered into adimeric fusion protein, whereby the fusion protein comprises two161P2F10B ligand binding domains linked to the Fc portion of a humanIgG, such as human IgG1. Such IgG portion can contain, for example, theC_(H)2 and C_(H)3 domains and the hinge region, but not the C_(H)1domain. Such dimeric fusion proteins are administered in soluble form topatients suffering from a cancer associated with the expression of161P2F10B, whereby the dimeric fusion protein specifically binds to161P2F10B and blocks 161P2F10B interaction with a binding partner. Suchdimeric fusion proteins are further combined into multimeric proteinsusing known antibody linking technologies.

XII.C.) Inhibition of 161P2F10B Transcription or Translation

The present invention also comprises various methods and compositionsfor inhibiting the transcription of the 161P2F10B gene. Similarly, theinvention also provides methods and compositions for inhibiting thetranslation of 161P2F10B mRNA into protein.

In one approach, a method of inhibiting the transcription of the161P2F10B gene comprises contacting the 161P2F10B gene with a 161P2F10Bantisense polynucleotide. In another approach, a method of inhibiting161P2F10B mRNA translation comprises contacting a 161P2F10B mRNA with anantisense polynucleotide. In another approach, a 161P2F10B specificribozyme is used to cleave a 161P2F10B message, thereby inhibitingtranslation. Such antisense and ribozyme based methods can also bedirected to the regulatory regions of the 161P2F10B gene, such as161P2F10B promoter and/or enhancer elements. Similarly, proteins capableof inhibiting a 161P2F10B gene transcription factor are used to inhibit161P2F10B mRNA transcription. The various polynucleotides andcompositions useful in the aforementioned methods have been describedabove. The use of antisense and ribozyme molecules to inhibittranscription and translation is well known in the art.

Other factors that inhibit the transcription of 161P2F10B by interferingwith 161P2F10B transcriptional activation are also useful to treatcancers expressing 161P2F10B. Similarly, factors that interfere with161P2F10B processing are useful to treat cancers that express 161P2F10B.Cancer treatment methods utilizing such factors are also within thescope of the invention.

XII.D.) General Considerations for Therapeutic Strategies

Gene transfer and gene therapy technologies can be used to delivertherapeutic polynucleotide molecules to tumor cells synthesizing161P2F10B (i.e., antisense, ribozyme, polynucleotides encodingintrabodies and other 161P2F10B inhibitory molecules). A number of genetherapy approaches are known in the art. Recombinant vectors encoding161P2F10B antisense polynucleotides, ribozymes, factors capable ofinterfering with 161P2F10B transcription, and so forth, can be deliveredto target tumor cells using such gene therapy approaches.

The above therapeutic approaches can be combined with any one of a widevariety of surgical, chemotherapy or radiation therapy regimens. Thetherapeutic approaches of the invention can enable the use of reduceddosages of chemotherapy (or other therapies) and/or less frequentadministration, an advantage for all patients and particularly for thosethat do not tolerate the toxicity of the chemotherapeutic agent well.

The anti-tumor activity of a particular composition (e.g., antisense,ribozyme, intrabody), or a combination of such compositions, can beevaluated using various in vitro and in vivo assay systems. In vitroassays that evaluate therapeutic activity include cell growth assays,soft agar assays and other assays indicative of tumor promotingactivity, binding assays capable of determining the extent to which atherapeutic composition will inhibit the binding of 161P2F10B to abinding partner, etc.

In vivo, the effect of a 161P2F10B therapeutic composition can beevaluated in a suitable animal model. For example, xenogenic prostatecancer models can be used, wherein human prostate cancer explants orpassaged xenograft tissues are introduced into immune compromisedanimals, such as nude or SCID mice (Klein et al., 1997, Nature Medicine3: 402-408). For example, PCT Patent Application WO98/16628 and U.S.Pat. No. 6,107,540 describe various xenograft models of human prostatecancer capable of recapitulating the development of primary tumors,micrometastasis, and the formation of osteoblastic metastasescharacteristic of late stage disease. Efficacy can be predicted usingassays that measure inhibition of tumor formation, tumor regression ormetastasis, and the like.

In vivo assays that evaluate the promotion of apoptosis are useful inevaluating therapeutic compositions. In one embodiment, xenografts fromtumor bearing mice treated with the therapeutic composition can beexamined for the presence of apoptotic foci and compared to untreatedcontrol xenograft-bearing mice. The extent to which apoptotic foci arefound in the tumors of the treated mice provides an indication of thetherapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16^(th) Edition, A. Osal., Ed.,1980).

Therapeutic formulations can be solubilized and administered via anyroute capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like. Apreferred formulation for intravenous injection comprises thetherapeutic composition in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. Therapeutic protein preparations can be lyophilized and stored assterile powders, preferably under vacuum, and then reconstituted inbacteriostatic water (containing for example, benzyl alcoholpreservative) or in sterile water prior to injection.

Dosages and administration protocols for the treatment of cancers usingthe foregoing methods will vary with the method and the target cancer,and will generally depend on a number of other factors appreciated inthe art.

XIII.) Identification, Characterization and Use of Modulators of161P2F10b

Methods to Identify and Use Modulators

In one embodiment, screening is performed to identify modulators thatinduce or suppress a particular expression profile, suppress or inducespecific pathways, preferably generating the associated phenotypethereby. In another embodiment, having identified differentiallyexpressed genes important in a particular state; screens are performedto identify modulators that alter expression of individual genes, eitherincrease or decrease. In another embodiment, screening is performed toidentify modulators that alter a biological function of the expressionproduct of a differentially expressed gene. Again, having identified theimportance of a gene in a particular state, screens are performed toidentify agents that bind and/or modulate the biological activity of thegene product.

In addition, screens are done for genes that are induced in response toa candidate agent. After identifying a modulator (one that suppresses acancer expression pattern leading to a normal expression pattern, or amodulator of a cancer gene that leads to expression of the gene as innormal tissue) a screen is performed to identify genes that arespecifically modulated in response to the agent. Comparing expressionprofiles between normal tissue and agent-treated cancer tissue revealsgenes that are not expressed in normal tissue or cancer tissue, but areexpressed in agent treated tissue, and vice versa. These agent-specificsequences are identified and used by methods described herein for cancergenes or proteins. In particular these sequences and the proteins theyencode are used in marking or identifying agent-treated cells. Inaddition, antibodies are raised against the agent-induced proteins andused to target novel therapeutics to the treated cancer tissue sample.

Modulator-Related Identification and Screening Assays:

Gene Expression-Related Assays

Proteins, nucleic acids, and antibodies of the invention are used inscreening assays. The cancer-associated proteins, antibodies, nucleicacids, modified proteins and cells containing these sequences are usedin screening assays, such as evaluating the effect of drug candidates ona “gene expression profile,” expression profile of polypeptides oralteration of biological function. In one embodiment, the expressionprofiles are used, preferably in conjunction with high throughputscreening techniques to allow monitoring for expression profile genesafter treatment with a candidate agent (e.g., Davis, G F, et al, J BiolScreen 7:69 (2002); Zlokarnik, et al., Science 279:84-8 (1998); Heid,Genome Res 6:986-94,1996).

The cancer proteins, antibodies, nucleic acids, modified proteins andcells containing the native or modified cancer proteins or genes areused in screening assays. That is, the present invention comprisesmethods for screening for compositions which modulate the cancerphenotype or a physiological function of a cancer protein of theinvention. This is done on a gene itself or by evaluating the effect ofdrug candidates on a “gene expression profile” or biological function.In one embodiment, expression profiles are used, preferably inconjunction with high throughput screening techniques to allowmonitoring after treatment with a candidate agent, see Zlokamik, supra.

A variety of assays are executed directed to the genes and proteins ofthe invention. Assays are run on an individual nucleic acid or proteinlevel. That is, having identified a particular gene as up regulated incancer, test compounds are screened for the ability to modulate geneexpression or for binding to the cancer protein of the invention.“Modulation” in this context includes an increase or a decrease in geneexpression. The preferred amount of modulation will depend on theoriginal change of the gene expression in normal versus tissueundergoing cancer, with changes of at least 10%, preferably 50%, morepreferably 100-300%, and in some embodiments 300-1000% or greater. Thus,if a gene exhibits a 4-fold increase in cancer tissue compared to normaltissue, a decrease of about four-fold is often desired; similarly, a10-fold decrease in cancer tissue compared to normal tissue a targetvalue of a 10-fold increase in expression by the test compound is oftendesired. Modulators that exacerbate the type of gene expression seen incancer are also useful, e.g., as an upregulated target in furtheranalyses.

The amount of gene expression is monitored using nucleic acid probes andthe quantification of gene expression levels, or, alternatively, a geneproduct itself is monitored, e.g., through the use of antibodies to thecancer protein and standard immunoassays. Proteomics and separationtechniques also allow for quantification of expression.

Expression Monitoring to Identify Compounds that Modify Gene Expression

In one embodiment, gene expression monitoring, i.e., an expressionprofile, is monitored simultaneously for a number of entities. Suchprofiles will typically involve one or more of the genes of FIG. 2. Inthis embodiment, e.g., cancer nucleic acid probes are attached tobiochips to detect and quantify cancer sequences in a particular cell.Alternatively, PCR can be used. Thus, a series, e.g., wells of amicrotiter plate, can be used with dispensed primers in desired wells. APCR reaction can then be performed and analyzed for each well.

Expression monitoring is performed to identify compounds that modify theexpression of one or more cancer-associated sequences, e.g., apolynucleotide sequence set out in FIG. 2. Generally, a test modulatoris added to the cells prior to analysis. Moreover, screens are alsoprovided to identify agents that modulate cancer, modulate cancerproteins of the invention, bind to a cancer protein of the invention, orinterfere with the binding of a cancer protein of the invention and anantibody or other binding partner.

In one embodiment, high throughput screening methods involve providing alibrary containing a large number of potential therapeutic compounds(candidate compounds). Such “combinatorial chemical libraries” are thenscreened in one or more assays to identify those library members(particular chemical species or subclasses) that display a desiredcharacteristic activity. The compounds thus identified can serve asconventional “lead compounds,” as compounds for screening, or astherapeutics.

In certain embodiments, combinatorial libraries of potential modulatorsare screened for an ability to bind to a cancer polypeptide or tomodulate activity. Conventionally, new chemical entities with usefulproperties are generated by identifying a chemical compound (called a“lead compound”) with some desirable property or activity, e.g.,inhibiting activity, creating variants of the lead compound, andevaluating the property and activity of those variant compounds. Often,high throughput screening (HTS) methods are employed for such ananalysis.

As noted above, gene expression monitoring is conveniently used to testcandidate modulators (e.g., protein, nucleic acid or small molecule).After the candidate agent has been added and the cells allowed toincubate for a period, the sample containing a target sequence to beanalyzed is, e.g., added to a biochip.

If required, the target sequence is prepared using known techniques. Forexample, a sample is treated to lyse the cells, using known lysisbuffers, electroporation, etc., with purification and/or amplificationsuch as PCR performed as appropriate. For example, an in vitrotranscription with labels covalently attached to the nucleotides isperformed. Generally, the nucleic acids are labeled with biotin-FITC orPE, or with cy3 or cy5.

The target sequence can be labeled with, e.g., a fluorescent, achemiluminescent, a chemical, or a radioactive signal, to provide ameans of detecting the target sequence's specific binding to a probe.The label also can be an enzyme, such as alkaline phosphatase orhorseradish peroxidase, which when provided with an appropriatesubstrate produces a product that is detected. Alternatively, the labelis a labeled compound or small molecule, such as an enzyme inhibitor,that binds but is not catalyzed or altered by the enzyme. The label alsocan be a moiety or compound, such as, an epitope tag or biotin whichspecifically binds to streptavidin. For the example of biotin, thestreptavidin is labeled as described above, thereby, providing adetectable signal for the bound target sequence. Unbound labeledstreptavidin is typically removed prior to analysis.

As will be appreciated by those in the art, these assays can be directhybridization assays or can comprise “sandwich assays”, which includethe use of multiple probes, as is generally outlined in U.S. Pat. Nos.5,681,702; 5,597,909; 5,545,730; 5,594,117; 5,591,584; 5,571,670;5,580,731; 5,571,670; 5,591,584; 5,624,802; 5,635,352; 5,594,118;5,359,100; 5,124,246; and 5,681,697. In this embodiment, in general, thetarget nucleic acid is prepared as outlined above, and then added to thebiochip comprising a plurality of nucleic acid probes, under conditionsthat allow the formation of a hybridization complex.

A variety of hybridization conditions are used in the present invention,including high, moderate and low stringency conditions as outlinedabove. The assays are generally run under stringency conditions whichallow formation of the label probe hybridization complex only in thepresence of target. Stringency can be controlled by altering a stepparameter that is a thermodynamic variable, including, but not limitedto, temperature, formamide concentration, salt concentration, chaotropicsalt concentration pH, organic solvent concentration, etc. Theseparameters may also be used to control non-specific binding, as isgenerally outlined in U.S. Pat. No. 5,681,697. Thus, it can be desirableto perform certain steps at higher stringency conditions to reducenon-specific binding.

The reactions outlined herein can be accomplished in a variety of ways.Components of the reaction can be added simultaneously, or sequentially,in different orders, with preferred embodiments outlined below. Inaddition, the reaction may include a variety of other reagents. Theseinclude salts, buffers, neutral proteins, e.g. albumin, detergents, etc.which can be used to facilitate optimal hybridization and detection,and/or reduce nonspecific or background interactions. Reagents thatotherwise improve the efficiency of the assay, such as proteaseinhibitors, nuclease inhibitors, anti-microbial agents, etc., may alsobe used as appropriate, depending on the sample preparation methods andpurity of the target. The assay data are analyzed to determine theexpression levels of individual genes, and changes in expression levelsas between states, forming a gene expression profile.

Biological Activity-Related Assays

The invention provides methods identify or screen for a compound thatmodulates the activity of a cancer-related gene or protein of theinvention. The methods comprise adding a test compound, as definedabove, to a cell comprising a cancer protein of the invention. The cellscontain a recombinant nucleic acid that encodes a cancer protein of theinvention. In another embodiment, a library of candidate agents istested on a plurality of cells.

In one aspect, the assays are evaluated in the presence or absence orprevious or subsequent exposure of physiological signals, e.g. hormones,antibodies, peptides, antigens, cytokines, growth factors, actionpotentials, pharmacological agents including chemotherapeutics,radiation, carcinogenics, or other cells (i.e., cell-cell contacts). Inanother example, the determinations are made at different stages of thecell cycle process. In this way, compounds that modulate genes orproteins of the invention are identified. Compounds with pharmacologicalactivity are able to enhance or interfere with the activity of thecancer protein of the invention. Once identified, similar structures areevaluated to identify critical structural features of the compound.

In one embodiment, a method of modulating (e.g., inhibiting) cancer celldivision is provided; the method comprises administration of a cancermodulator. In another embodiment, a method of modulating (e.g.,inhibiting) cancer is provided; the method comprises administration of acancer modulator. In a further embodiment, methods of treating cells orindividuals with cancer are provided; the method comprisesadministration of a cancer modulator.

In one embodiment, a method for modulating the status of a cell thatexpresses a gene of the invention is provided. As used herein statuscomprises such art-accepted parameters such as growth, proliferation,survival, function, apoptosis, senescence, location, enzymatic activity,signal transduction, etc. of a cell. In one embodiment, a cancerinhibitor is an antibody as discussed above. In another embodiment, thecancer inhibitor is an antisense molecule. A variety of cell growth,proliferation, and metastasis assays are known to those of skill in theart, as described herein.

High Throughput Screening to Identify Modulators

The assays to identify suitable modulators are amenable to highthroughput screening. Preferred assays thus detect enhancement orinhibition of cancer gene transcription, inhibition or enhancement ofpolypeptide expression, and inhibition or enhancement of polypeptideactivity.

In one embodiment, modulators evaluated in high throughput screeningmethods are proteins, often naturally occurring proteins or fragments ofnaturally occurring proteins. Thus, e.g., cellular extracts containingproteins, or random or directed digests of proteinaceous cellularextracts, are used. In this way, libraries of proteins are made forscreening in the methods of the invention. Particularly preferred inthis embodiment are libraries of bacterial, fungal, viral, and mammalianproteins, with the latter being preferred, and human proteins beingespecially preferred. Particularly useful test compound will be directedto the class of proteins to which the target belongs, e.g., substratesfor enzymes, or ligands and receptors.

Use of Soft Agar Growth and Colony Formation to Identify andCharacterize Modulators

Normal cells require a solid substrate to attach and grow. When cellsare transformed, they lose this phenotype and grow detached from thesubstrate. For example, transformed cells can grow in stirred suspensionculture or suspended in semi-solid media, such as semi-solid or softagar. The transformed cells, when transfected with tumor suppressorgenes, can regenerate normal phenotype and once again require a solidsubstrate to attach to and grow. Soft agar growth or colony formation inassays are used to identify modulators of cancer sequences, which whenexpressed in host cells, inhibit abnormal cellular proliferation andtransformation. A modulator reduces or eliminates the host cells'ability to grow suspended in solid or semisolid media, such as agar.

Techniques for soft agar growth or colony formation in suspension assaysare described in Freshney, Culture of Animal Cells a Manual of BasicTechnique (3rd ed., 1994). See also, the methods section of Garkavtsevet al. (1996), supra.

Evaluation of Contact Inhibition and Growth Density Limitation toIdentify and Characterize Modulators

Normal cells typically grow in a flat and organized pattern in cellculture until they touch other cells. When the cells touch one another,they are contact inhibited and stop growing. Transformed cells, however,are not contact inhibited and continue to grow to high densities indisorganized foci. Thus, transformed cells grow to a higher saturationdensity than corresponding normal cells. This is detectedmorphologically by the formation of a disoriented monolayer of cells orcells in foci. Alternatively, labeling index with (³H)-thymidine atsaturation density is used to measure density limitation of growth,similarly an MTT or Alamar blue assay will reveal proliferation capacityof cells and the the ability of modulators to affect same. See Freshney(1994), supra. Transformed cells, when transfected with tumor suppressorgenes, can regenerate a normal phenotype and become contact inhibitedand would grow to a lower density.

In this assay, labeling index with ³H)-thymidine at saturation densityis a preferred method of measuring density limitation of growth.Transformed host cells are transfected with a cancer-associated sequenceand are grown for 24 hours at saturation density in non-limiting mediumconditions. The percentage of cells labeling with (³H)-thymidine isdetermined by incorporated cpm.

Contact independent growth is used to identify modulators of cancersequences, which had led to abnormal cellular proliferation andtransformation. A modulator reduces or eliminates contact independentgrowth, and returns the cells to a normal phenotype.

Evaluation of Growth Factor or Serum Dependence to Identify andCharacterize Modulators

Transformed cells have lower serum dependence than their normalcounterparts (see, e.g., Temin, J. Natl. Cancer Inst. 37:167-175 (1966);Eagle et al., J. Exp. Med 131:836-879 (1970)); Freshney, supra. This isin part due to release of various growth factors by the transformedcells. The degree of growth factor or serum dependence of transformedhost cells can be compared with that of control. For example, growthfactor or serum dependence of a cell is monitored in methods to identifyand characterize compounds that modulate cancer-associated sequences ofthe invention.

Use of Tumor-Specific Marker Levels to Identify and CharacterizeModulators

Tumor cells release an increased amount of certain factors (hereinafter“tumor specific markers”) than their normal counterparts. For example,plasminogen activator (PA) is released from human glioma at a higherlevel than from normal brain cells (see, e.g., Gullino, Angiogenesis,Tumor Vascularization, and Potential Interference with Tumor Growth, inBiological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985)).Similarly, Tumor Angiogenesis Factor (TAF) is released at a higher levelin tumor cells than their normal counterparts. See, e.g., Folkman,Angiogenesis and Cancer, Sem Cancer Biol. (1992)), while bFGF isreleased from endothelial tumors (Ensoli, B et al). Various techniqueswhich measure the release of these factors are described in Freshney(1994), supra. Also, see, Unkless et al., J. Biol. Chem. 249:4295-4305(1974); Strickland & Beers, J. Biol. Chem. 251:5694-5702 (1976); Whur etal., Br. J. Cancer 42:305 312 (1980); Gullino, Angiogenesis, TumorVascularization, and Potential Interference with Tumor Growth, inBiological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985);Freshney, Anticancer Res. 5:111-130 (1985). For example, tumor specificmarker levels are monitored in methods to identify and characterizecompounds that modulate cancer-associated sequences of the invention.

Invasiveness into Matrigel to Identify and Characterize Modulators

The degree of invasiveness into Matrigel or an extracellular matrixconstituent can be used as an assay to identify and characterizecompounds that modulate cancer associated sequences. Tumor cells exhibita positive correlation between malignancy and invasiveness of cells intoMatrigel or some other extracellular matrix constituent. In this assay,tumorigenic cells are typically used as host cells. Expression of atumor suppressor gene in these host cells would decrease invasiveness ofthe host cells. Techniques described in Cancer Res. 1999; 59:6010;Freshney (1994), supra, can be used. Briefly, the level of invasion ofhost cells is measured by using filters coated with Matrigel or someother extracellular matrix constituent. Penetration into the gel, orthrough to the distal side of the filter, is rated as invasiveness, andrated histologically by number of cells and distance moved, or byprelabeling the cells with ¹²⁵1 and counting the radioactivity on thedistal side of the filter or bottom of the dish. See, e.g., Freshney(1984), supra.

Evaluation of Tumor Growth In Vivo to Identify and CharacterizeModulators

Effects of cancer-associated sequences on cell growth are tested intransgenic or immune-suppressed organisms. Transgenic organisms areprepared in a variety of art-accepted ways. For example, knock-outtransgenic organisms, e.g., mammals such as mice, are made, in which acancer gene is disrupted or in which a cancer gene is inserted.Knock-out transgenic mice are made by insertion of a marker gene orother heterologous gene into the endogenous cancer gene site in themouse genome via homologous recombination. Such mice can also be made bysubstituting the endogenous cancer gene with a mutated version of thecancer gene, or by mutating the endogenous cancer gene, e.g., byexposure to carcinogens.

To prepare transgenic chimeric animals, e.g., mice, a DNA construct isintroduced into the nuclei of embryonic stem cells. Cells containing thenewly engineered genetic lesion are injected into a host mouse embryo,which is re-implanted into a recipient female. Some of these embryosdevelop into chimeric mice that possess germ cells some of which arederived from the mutant cell line. Therefore, by breeding the chimericmice it is possible to obtain a new line of mice containing theintroduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288(1989)). Chimeric mice can be derived according to U.S. Pat. No.6,365,797, issued 2 Apr. 2002; U.S. Pat. No. 6,107,540 issued 22 Aug.2000; Hogan et al., Manipulating the Mouse Embryo: A laboratory Manual,Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and EmbryonicStem Cells: A Practical Approach, Robertson, ed., IRL Press, Washington,D.C., (1987).

Alternatively, various immune-suppressed or immune-deficient hostanimals can be used. For example, a genetically athymic “nude” mouse(see, e.g., Giovanella et al., J. Natl. Cancer Inst. 52:921 (1974)), aSCID mouse, a thymectornized mouse, or an irradiated mouse (see, e.g.,Bradley et al., Br. J. Cancer 38:263 (1978); Selby et al., Br. J. Cancer41:52 (1980)) can be used as a host. Transplantable tumor cells(typically about 106 cells) injected into isogenic hosts produceinvasive tumors in a high proportion of cases, while normal cells ofsimilar origin will not. In hosts which developed invasive tumors, cellsexpressing cancer-associated sequences are injected subcutaneously ororthotopically. Mice are then separated into groups, including controlgroups and treated experimental groups) e.g. treated with a modulator).After a suitable length of time, preferably 4-8 weeks, tumor growth ismeasured (e.g., by volume or by its two largest dimensions, or weight)and compared to the control. Tumors that have statistically significantreduction (using, e.g., Student's T test) are said to have inhibitedgrowth.

In Vitro Assays to Identify and Characterize Modulators

Assays to identify compounds with modulating activity can be performedin vitro. For example, a cancer polypeptide is first contacted with apotential modulator and incubated for a suitable amount of time, e.g.,from 0.5 to 48 hours. In one embodiment, the cancer polypeptide levelsare determined in vitro by measuring the level of protein or mRNA. Thelevel of protein is measured using immunoassays such as Westernblotting, ELISA and the like with an antibody that selectively binds tothe cancer polypeptide or a fragment thereof. For measurement of mRNA,amplification, e.g., using PCR, LCR, or hybridization assays, e.g.,Northern hybridization, RNAse protection, dot blotting, are preferred.The level of protein or mRNA is detected using directly or indirectlylabeled detection agents, e.g., fluorescently or radioactively labelednucleic acids, radioactively or enzymatically labeled antibodies, andthe like, as described herein.

Alternatively, a reporter gene system can be devised using a cancerprotein promoter operably linked to a reporter gene such as luciferase,green fluorescent protein, CAT, or P-gal. The reporter construct istypically transfected into a cell. After treatment with a potentialmodulator, the amount of reporter gene transcription, translation, oractivity is measured according to standard techniques known to those ofskill in the art (Davis G F, supra; Gonzalez, J. & Negulescu, P. Curr.Opin. Biotechnol. 1998: 9:624).

As outlined above, in vitro screens are done on individual genes andgene products. That is, having identified a particular differentiallyexpressed gene as important in a particular state, screening ofmodulators of the expression of the gene or the gene product itself isperformed.

In one embodiment, screening for modulators of expression of specificgene(s) is performed. Typically, the expression of only one or a fewgenes is evaluated. In another embodiment, screens are designed to firstfind compounds that bind to differentially expressed proteins. Thesecompounds are then evaluated for the ability to modulate differentiallyexpressed activity. Moreover, once initial candidate compounds areidentified, variants can be further screened to better evaluatestructure activity relationships.

Binding Assays to Identify and Characterize Modulators

In binding assays in accordance with the invention, a purified orisolated gene product of the invention is generally used. For example,antibodies are generated to a protein of the invention, and immunoassaysare run to determine the amount and/or location of protein.Alternatively, cells comprising the cancer proteins are used in theassays.

Thus, the methods comprise combining a cancer protein of the inventionand a candidate compound such as a ligand, and determining the bindingof the compound to the cancer protein of the invention. Preferredembodiments utilize the human cancer protein; animal models of humandisease of can also be developed and used. Also, other analogousmammalian proteins also can be used as appreciated by those of skill inthe art. Moreover, in some embodiments variant or derivative cancerproteins are used.

Generally, the cancer protein of the invention, or the ligand, isnon-diffusibly bound to an insoluble support. The support can, e.g., beone having isolated sample receiving areas (a microtiter plate, anarray, etc.). The insoluble supports can be made of any composition towhich the compositions can be bound, is readily separated from solublematerial, and is otherwise compatible with the overall method ofscreening. The surface of such supports can be solid or porous and ofany convenient shape.

Examples of suitable insoluble supports include microtiter plates,arrays, membranes and beads. These are typically made of glass, plastic(e.g., polystyrene), polysaccharide, nylon, nitrocellulose, or Teflon™,etc. Microtiter plates and arrays are especially convenient because alarge number of assays can be carried out simultaneously, using smallamounts of reagents and samples. The particular manner of binding of thecomposition to the support is not crucial so long as it is compatiblewith the reagents and overall methods of the invention, maintains theactivity of the composition and is nondiffusable. Preferred methods ofbinding include the use of antibodies which do not sterically blockeither the ligand binding site or activation sequence when attaching theprotein to the support, direct binding to “sticky” or ionic supports,chemical crosslinking, the synthesis of the protein or agent on thesurface, etc. Following binding of the protein or ligand/binding agentto the support, excess unbound material is removed by washing. Thesample receiving areas may then be blocked through incubation withbovine serum albumin (BSA), casein or other innocuous protein or othermoiety.

Once a cancer protein of the invention is bound to the support, and atest compound is added to the assay. Alternatively, the candidatebinding agent is bound to the support and the cancer protein of theinvention is then added. Binding agents include specific antibodies,non-natural binding agents identified in screens of chemical libraries,peptide analogs, etc.

Of particular interest are assays to identify agents that have a lowtoxicity for human cells. A wide variety of assays can be used for thispurpose, including proliferation assays, cAMP assays, labeled in vitroprotein-protein binding assays, electrophoretic mobility shift assays,immunoassays for protein binding, functional assays (phosphorylationassays, etc.) and the like.

A determination of binding of the test compound (ligand, binding agent,modulator, etc.) to a cancer protein of the invention can be done in anumber of ways. The test compound can be labeled, and binding determineddirectly, e.g., by attaching all or a portion of the cancer protein ofthe invention to a solid support, adding a labeled candidate compound(e.g., a fluorescent label), washing off excess reagent, and determiningwhether the label is present on the solid support. Various blocking andwashing steps can be utilized as appropriate.

In certain embodiments, only one of the components is labeled, e.g., aprotein of the invention or ligands labeled. Alternatively, more thanone component is labeled with different labels, e.g., I125, for theproteins and a fluorophor for the compound. Proximity reagents, e.g.,quenching or energy transfer reagents are also useful.

Competitive Binding to Identify and Characterize Modulators

In one embodiment, the binding of the “test compound” is determined bycompetitive binding assay with a “competitor.” The competitor is abinding moiety that binds to the target molecule (e.g., a cancer proteinof the invention). Competitors include compounds such as antibodies,peptides, binding partners, ligands, etc. Under certain circumstances,the competitive binding between the test compound and the competitordisplaces the test compound. In one embodiment, the test compound islabeled. Either the test compound, the competitor, or both, is added tothe protein for a time sufficient to allow binding. Incubations areperformed at a temperature that facilitates optimal activity, typicallybetween four and 40° C. Incubation periods are typically optimized,e.g., to facilitate rapid high throughput screening; typically betweenzero and one hour will be sufficient. Excess reagent is generallyremoved or washed away. The second component is then added, and thepresence or absence of the labeled component is followed, to indicatebinding.

In one embodiment, the competitor is added first, followed by the testcompound. Displacement of the competitor is an indication that the testcompound is binding to the cancer protein and thus is capable of bindingto, and potentially modulating, the activity of the cancer protein. Inthis embodiment, either component can be labeled. Thus, e.g., if thecompetitor is labeled, the presence of label in the post-test compoundwash solution indicates displacement by the test compound.Alternatively, if the test compound is labeled, the presence of thelabel on the support indicates displacement.

In an alternative embodiment, the test compound is added first, withincubation and washing, followed by the competitor. The absence ofbinding by the competitor indicates that the test compound binds to thecancer protein with higher affinity than the competitor. Thus, if thetest compound is labeled, the presence of the label on the support,coupled with a lack of competitor binding, indicates that the testcompound binds to and thus potentially modulates the cancer protein ofthe invention.

Accordingly, the competitive binding methods comprise differentialscreening to identity agents that are capable of modulating the activityof the cancer proteins of the invention. In this embodiment, the methodscomprise combining a cancer protein and a competitor in a first sample.A second sample comprises a test compound, the cancer protein, and acompetitor. The binding of the competitor is determined for bothsamples, and a change, or difference in binding between the two samplesindicates the presence of an agent capable of binding to the cancerprotein and potentially modulating its activity. That is, if the bindingof the competitor is different in the second sample relative to thefirst sample, the agent is capable of binding to the cancer protein.

Alternatively, differential screening is used to identify drugcandidates that bind to the native cancer protein, but cannot bind tomodified cancer proteins. For example the structure of the cancerprotein is modeled and used in rational drug design to synthesize agentsthat interact with that site, agents which generally do not bind tosite-modified proteins. Moreover, such drug candidates that affect theactivity of a native cancer protein are also identified by screeningdrugs for the ability to either enhance or reduce the activity of suchproteins.

Positive controls and negative controls can be used in the assays.Preferably control and test samples are performed in at least triplicateto obtain statistically significant results. Incubation of all samplesoccurs for a time sufficient to allow for the binding of the agent tothe protein. Following incubation, samples are washed free ofnon-specifically bound material and the amount of bound, generallylabeled agent determined For example, where a radiolabel is employed,the samples can be counted in a scintillation counter to determine theamount of bound compound.

A variety of other reagents can be included in the screening assays.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc. which are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Alsoreagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.,can be used. The mixture of components is added in an order thatprovides for the requisite binding.

Use of Polynucleotides to Down-Regulate or Inhibit a Protein of theInvention.

Polynucleotide modulators of cancer can be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand-binding molecule, as described in WO 91/04753. Suitableligand-binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell. Alternatively, a polynucleotide modulator ofcancer can be introduced into a cell containing the target nucleic acidsequence, e.g., by formation of a polynucleotide-lipid complex, asdescribed in WO 90/10448. It is understood that the use of antisensemolecules or knock out and knock in models may also be used in screeningassays as discussed above, in addition to methods of treatment.

Inhibitory and Antisense Nucleotides

In certain embodiments, the activity of a cancer-associated protein isdown-regulated, or entirely inhibited, by the use of antisensepolynucleotide or inhibitory small nuclear RNA (snRNA), i.e., a nucleicacid complementary to, and which can preferably hybridize specificallyto, a coding mRNA nucleic acid sequence, e.g., a cancer protein of theinvention, mRNA, or a subsequence thereof. Binding of the antisensepolynucleotide to the mRNA reduces the translation and/or stability ofthe mRNA.

In the context of this invention, antisense polynucleotides can comprisenaturally occurring nucleotides, or synthetic species formed fromnaturally occurring subunits or their close homologs. Antisensepolynucleotides may also have altered sugar moieties or inter-sugarlinkages. Exemplary among these are the phosphorothioate and othersulfur containing species which are known for use in the art. Analogsare comprised by this invention so long as they function effectively tohybridize with nucleotides of the invention. See, e.g., IsisPharmaceuticals, Carlsbad, Calif.; Sequitor, Inc., Natick, Mass.

Such antisense polynucleotides can readily be synthesized usingrecombinant means, or can be synthesized in vitro. Equipment for suchsynthesis is sold by several vendors, including Applied Biosystems. Thepreparation of other oligonucleotides such as phosphorothioates andalkylated derivatives is also well known to those of skill in the art.

Antisense molecules as used herein include antisense or senseoligonucleotides. Sense oligonucleotides can, e.g., be employed to blocktranscription by binding to the anti-sense strand. The antisense andsense oligonucleotide comprise a single stranded nucleic acid sequence(either RNA or DNA) capable of binding to target mRNA (sense) or DNA(antisense) sequences for cancer molecules. Antisense or senseoligonucleotides, according to the present invention, comprise afragment generally at least about 12 nucleotides, preferably from about12 to 30 nucleotides. The ability to derive an antisense or a senseoligonucleotide, based upon a cDNA sequence encoding a given protein isdescribed in, e.g., Stein & Cohen (Cancer Res. 48:2659 (1988 and van derKrol et al. (BioTechniques 6:958 (1988)).

Ribozymes

In addition to antisense polynucleotides, ribozymes can be used totarget and inhibit transcription of cancer-associated nucleotidesequences. A ribozyme is an RNA molecule that catalytically cleavesother RNA molecules. Different kinds of ribozymes have been described,including group I ribozymes, hammerhead ribozymes, hairpin ribozymes,RNase P, and axhead ribozymes (see, e.g., Castanotto et al., Adv. inPharmacology 25: 289-317 (1994) for a general review of the propertiesof different ribozymes).

The general features of hairpin ribozymes are described, e.g., in Hampelet al., Nucl. Acids Res. 18:299-304 (1990); European Patent PublicationNo. 0360257; U.S. Pat. No. 5,254,678. Methods of preparing are wellknown to those of skill in the art (see, e.g., WO 94/26877; Ojwang etal., Proc. Natl. Acad. Sci. USA 90:6340-6344 (1993); Yamada et al.,Human Gene Therapy 1:39-45 (1994); Leavitt et al., Proc. Natl. Acad Sci.USA 92:699-703 (1995); Leavitt et al., Human Gene Therapy 5: 1151-120(1994); and Yamada et al., Virology 205: 121-126 (1994)).

Use of Modulators in Phenotypic Screening

In one embodiment, a test compound is administered to a population ofcancer cells, which have an associated cancer expression profile. By“administration” or “contacting” herein is meant that the modulator isadded to the cells in such a manner as to allow the modulator to actupon the cell, whether by uptake and intracellular action, or by actionat the cell surface. In some embodiments, a nucleic acid encoding aproteinaceous agent (i.e., a peptide) is put into a viral construct suchas an adenoviral or retroviral construct, and added to the cell, suchthat expression of the peptide agent is accomplished, e.g., PCTUS97/01019. Regulatable gene therapy systems can also be used. Once themodulator has been administered to the cells, the cells are washed ifdesired and are allowed to incubate under preferably physiologicalconditions for some period. The cells are then harvested and a new geneexpression profile is generated. Thus, e.g., cancer tissue is screenedfor agents that modulate, e.g., induce or suppress, the cancerphenotype. A change in at least one gene, preferably many, of theexpression profile indicates that the agent has an effect on canceractivity. Similarly, altering a biological function or a signalingpathway is indicative of modulator activity. By defining such asignature for the cancer phenotype, screens for new drugs that alter thephenotype are devised. With this approach, the drug target need not beknown and need not be represented in the original gene/proteinexpression screening platform, nor does the level of transcript for thetarget protein need to change. The modulator inhibiting function willserve as a surrogate marker

As outlined above, screens are done to assess genes or gene products.That is, having identified a particular differentially expressed gene asimportant in a particular state, screening of modulators of either theexpression of the gene or the gene product itself is performed.

Use of Modulators to Affect Peptides of the Invention

Measurements of cancer polypeptide activity, or of the cancer phenotypeare performed using a variety of assays. For example, the effects ofmodulators upon the function of a cancer polypeptide(s) are measured byexamining parameters described above. A physiological change thataffects activity is used to assess the influence of a test compound onthe polypeptides of this invention. When the functional outcomes aredetermined using intact cells or animals, a variety of effects can beassesses such as, in the case of a cancer associated with solid tumors,tumor growth, tumor metastasis, neovascularization, hormone release,transcriptional changes to both known and uncharacterized geneticmarkers (e.g., by Northern blots), changes in cell metabolism such ascell growth or pH changes, and changes in intracellular secondmessengers such as cGNIP.

Methods of Identifying Characterizing Cancer-Associated Sequences

Expression of various gene sequences is correlated with cancer.Accordingly, disorders based on mutant or variant cancer genes aredetermined In one embodiment, the invention provides methods foridentifying cells containing variant cancer genes, e.g., determining thepresence of, all or part, the sequence of at least one endogenous cancergene in a cell. This is accomplished using any number of sequencingtechniques. The invention comprises methods of identifying the cancergenotype of an individual, e.g., determining all or part of the sequenceof at least one gene of the invention in the individual. This isgenerally done in at least one tissue of the individual, e.g., a tissueset forth in Table I, and may include the evaluation of a number oftissues or different samples of the same tissue. The method may includecomparing the sequence of the sequenced gene to a known cancer gene,i.e., a wild-type gene to determine the presence of family members,homologies, mutations or variants. The sequence of all or part of thegene can then be compared to the sequence of a known cancer gene todetermine if any differences exist. This is done using any number ofknown homology programs, such as BLAST, Bestfit, etc. The presence of adifference in the sequence between the cancer gene of the patient andthe known cancer gene correlates with a disease state or a propensityfor a disease state, as outlined herein.

In a preferred embodiment, the cancer genes are used as probes todetermine the number of copies of the cancer gene in the genome. Thecancer genes are used as probes to determine the chromosomallocalization of the cancer genes. Information such as chromosomallocalization finds use in providing a diagnosis or prognosis inparticular when chromosomal abnormalities such as translocations, andthe like are identified in the cancer gene locus.

XIV.) Kits/Articles of Manufacture

For use in the diagnostic and therapeutic applications described herein,kits are also within the scope of the invention. Such kits can comprisea carrier, package or container that is compartmentalized to receive oneor more containers such as vials, tubes, and the like, each of thecontainer(s) comprising one of the separate elements to be used in themethod. For example, the container(s) can comprise a probe that is orcan be detectably labeled. Such probe can be an antibody orpolynucleotide specific for a FIG. 2-related protein or a FIG. 2 gene ormessage, respectively. Where the method utilizes nucleic acidhybridization to detect the target nucleic acid, the kit can also havecontainers containing nucleotide(s) for amplification of the targetnucleic acid sequence and/or a container comprising a reporter-means,such as a biotin-binding protein, such as avidin or streptavidin, boundto a reporter molecule, such as an enzymatic, florescent, orradioisotope label. The kit can include all or part of the amino acidsequences in FIG. 2 or FIG. 3 or analogs thereof, or a nucleic acidmolecules that encodes such amino acid sequences.

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including buffers, diluents,filters, needles, syringes; carrier, package, container, vial and/ortube labels listing contents and/or instructions for use, and packageinserts with instructions for use.

A label can be present on the container to indicate that the compositionis used for a specific therapy or non-therapeutic application, such as adiagnostic or laboratory application, and can also indicate directionsfor either in vivo or in vitro use, such as those described herein.Directions and or other information can also be included on an insert(s)or label(s) which is included with or on the kit.

The terms “kit” and “article of manufacture” can be used as synonyms.

In another embodiment of the invention, an article(s) of manufacturecontaining compositions, such as amino acid sequence(s), smallmolecule(s), nucleic acid sequence(s), and/or antibody(s), e.g.,materials useful for the diagnosis, prognosis, prophylaxis and/ortreatment of neoplasias of tissues such as those set forth in Table I isprovided. The article of manufacture typically comprises at least onecontainer and at least one label. Suitable containers include, forexample, bottles, vials, syringes, and test tubes. The containers can beformed from a variety of materials such as glass or plastic. Thecontainer can hold amino acid sequence(s), small molecule(s), nucleicacid sequence(s), and/or antibody(s), in one embodiment the containerholds a polynucleotide for use in examining the mRNA expression profileof a cell, together with reagents used for this purpose.

The container can alternatively hold a composition which is effectivefor treating, diagnosis, prognosing or prophylaxing a condition and canhave a sterile access port (for example the container can be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). The active agents in the composition canbe an antibody capable of specifically binding 161P2F10B and modulatingthe function of 161P2F10B.

The label can be on or associated with the container. A label a can beon a container when letters, numbers or other characters forming thelabel are molded or etched into the container itself; a label can beassociated with a container when it is present within a receptacle orcarrier that also holds the container, e.g., as a package insert. Thelabel can indicate that the composition is used for diagnosing,treating, prophylaxing or prognosing a condition, such as a neoplasia ofa tissue set forth in Table I. The article of manufacture can furthercomprise a second container comprising a pharmaceutically-acceptablebuffer, such as phosphate-buffered saline, Ringer's solutionand/ordextrose solution. It can further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, stirrers, needles, syringes, and/or packageinserts with indications and/or instructions for use.

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the several examples that follow, none of which are intendedto limit the scope of the invention.

Example 1 SSH-Generated Isolation of cDNA Fragment of the STEAP Gene

To isolate genes that are over-expressed in kidney cancer we used theSuppression Subtractive Hybridization (SSH) procedure using cDNA derivedfrom kidney cancer patient tissues.

The 161P2F10B SSH cDNA sequence was derived from a subtractionconsisting of a kidney cancer minus normal kidney and a mixture of 9normal tissues: stomach, skeletal muscle, lung, brain, liver, kidney,pancreas, small intestine and heart. By RT-PCR, the 161P2F10B cDNA wasidentified as highly expressed in kidney cancer pool, with lowerexpression detected in prostate cancer xenograft pool, prostate cancerpool, colon cancer pool, lung cancer pool, ovary cancer pool, breastcancer pool, metastasis cancer pool, pancreas cancer pool, 2 differentprostate cancer metastasis to lymph node, VP1 and VP2. (FIG. 14).

The 161P2F10B SSH cDNA sequence of 182 by matches the cDNA for

phosphodiesterase I/nucleotide pyrophosphatase 3 (PDNP3). Thefull-length 161P2F10B cDNA and ORF are described in FIG. 2 with theprotein sequence listed in FIG. 3.

Materials and Methods

RNA Isolation:

Tumor tissues were homogenized in Trizol reagent (Life Technologies,Gibco BRL) using 10 ml/g tissue or 10 ml/10⁸ cells to isolate total RNA.Poly A RNA was purified from total RNA using Qiagen's Oligotex mRNA Miniand Midi kits. Total and mRNA were quantified by spectrophotometricanalysis (O.D. 260/280 nm) and analyzed by gel electrophoresis.

Oligonucleotides:

The following HPLC purified oligonucleotides were used.

DPNCDN (cDNA Synthesis Primer):

5′TTTTGATCAAGCTT₃₀3′ (SEQ ID NO: 29)

Adaptor 1:

(SEQ ID NO: 30) 5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′ (SEQ IDNO: 31) 3′GGCCCGTCCTAG5′

Adaptor 2:

(SEQ ID NO: 32) 5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ (SEQ ID NO:33) 3′CGGCTCCTAG5′

PCR Primer 1:

5′CTAATACGACTCACTATAGGGC3′ (SEQ ID NO: 34)

Nested Primer (NP)1:

5′TCGAGCGGCCGCCCGGGCAGGA3′ (SEQ ID NO: 35)

Nested Primer (NP)2:

5′AGCGTGGTCGCGGCCGAGGA3′ (SEQ ID NO: 36)

Suppression Subtractive Hybridization:

Suppression Subtractive Hybridization (SSH) was used to identify cDNAscorresponding to genes that may be differentially expressed in prostatecancer. The SSH reaction utilized cDNA from kidney cancer patientspecimens. The gene 161P2F10B was derived from kidney cancer patienttissues minus normal kidney and a mixture of 9 normal tissues: stomach,skeletal muscle, lung, brain, liver, kidney, pancreas, small intestineand heart. The SSH DNA sequence (FIG. 1) was identified.

The cDNA derived from kidney cancer patient tissues was used as thesource of the “driver” cDNA, while the cDNA from normal tissues was usedas the source of the “tester” cDNA. Double stranded cDNAs correspondingto tester and driver cDNAs were synthesized from 2 μg of poly(A)⁺ RNAisolated from the relevant tissue, as described above, using CLONTECH'sPCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotide DPNCDN asprimer. First- and second-strand synthesis were carried out as describedin the Kit's user manual protocol (CLONTECH Protocol No. PT1117-1,Catalog No. K1804-1). The resulting cDNA was digested with Dpn II for 3hrs at 37° C. Digested cDNA was extracted with phenol/chloroform (1:1)and ethanol precipitated.

Tester cDNA was generated by diluting 1 μl of Dpn II digested cDNA fromthe relevant tissue source (see above) (400 ng) in 5 μl of water. Thediluted cDNA (2 μl, 160 ng) was then ligated to 2 μl A of Adaptor 1 andAdaptor 2 (10 μM), in separate ligation reactions, in a total volume of10 μl at 16° C. overnight, using 400 u of T4 DNA ligase (CLONTECH).Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72° C.for 5 min

The first hybridization was performed by adding 1.5 μl (600 ng) ofdriver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1-and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, thesamples were overlaid with mineral oil, denatured in an MJ Researchthermal cycler at 98° C. for 1.5 minutes, and then were allowed tohybridize for 8 hrs at 68° C. The two hybridizations were then mixedtogether with an additional 1 μl of fresh denatured driver cDNA and wereallowed to hybridize overnight at 68° C. The second hybridization wasthen diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA,heated at 70° C. for 7 min and stored at −20° C.

PCR Amplification, Cloning and Sequencing of Gene Fragments GeneratedFrom SSH:

To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 μl of thediluted final hybridization mix was added to 1 μl of PCR primer 1 (10μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10×reaction buffer (CLONTECH) and0.5 μl 50×Advantage cDNA polymerase Mix (CLONTECH) in a final volume of25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5mM, 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C. for30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions wereperformed for each experiment. The products were pooled and diluted 1:10with water. For the secondary PCR reaction, 1 μl from the pooled anddiluted primary PCR reaction was added to the same reaction mix as usedfor PCR 1, except that primers NP1 and NP2 (10 μM) were used instead ofPCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10sec, 68° C. for 30 sec, and 72° C. for 1.5 minutes. The PCR productswere analyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloningkit (Invitrogen). Transformed E. coli were subjected to blue/white andampicillin selection. White colonies were picked and arrayed into 96well plates and were grown in liquid culture overnight. To identifyinserts, PCR amplification was performed on 1 ml of bacterial cultureusing the conditions of PCR1 and NP1 and NP2 as primers. PCR productswere analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format.Plasmid DNA was prepared, sequenced, and subjected to nucleic acidhomology searches of the GenBank, dBest, and NCI-CGAP databases.

RT-PCR Expression Analysis:

First strand cDNAs can be generated from 1 μg of mRNA with oligo(dT)12-18 priming using the Gibco-BRL Superscript Preamplificationsystem. The manufacturer's protocol was used which included anincubation for 50 min at 42° C. with reverse transcriptase followed byRNAse H treatment at 37° C. for 20 min. After completing the reaction,the volume can be increased to 200 μl with water prior to normalization.First strand cDNAs from 16 different normal human tissues can beobtained from Clontech.

Normalization of the first strand cDNAs from multiple tissues wasperformed by using the primers 5′ atatcgccgcgctcgtcgtcgacaa3′ (SEQ IDNO: 37) and 5′ agccacacgcagctcattgtagaagg 3′ (SEQ ID NO: 38) to amplifyβ-actin. First strand cDNA (5 μl) were amplified in a total volume of 50μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCR buffer (Clontech,10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, pH8.3) and 1× Klentaq DNApolymerase (Clontech). Five μl of the PCR reaction can be removed at 18,20, and 22 cycles and used for agarose gel electrophoresis. PCR wasperformed using an MJ Research thermal cycler under the followingconditions: Initial denaturation can be at 94° C. for 15 sec, followedby a 18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 min, 72° C.for 5 sec. A final extension at 72° C. was carried out for 2 min. Afteragarose gel electrophoresis, the band intensities of the 283 by β-actinbands from multiple tissues were compared by visual inspection. Dilutionfactors for the first strand cDNAs were calculated to result in equalβ-actin band intensities in all tissues after 22 cycles of PCR. Threerounds of normalization can be required to achieve equal bandintensities in all tissues after 22 cycles of PCR.

To determine expression levels of the 161P2F10B gene, 5 μl of normalizedfirst strand cDNA were analyzed by PCR using 26, and 30 cycles ofamplification. Semi-quantitative expression analysis can be achieved bycomparing the PCR products at cycle numbers that give light bandintensities.

A typical RT-PCR expression analysis is shown in FIG. 14. RT-PCRexpression analysis was performed on first strand cDNAs generated usingpools of tissues from multiple samples. The cDNAs were shown to benormalized using beta-actin PCR. Strong expression of 161P2F10B wasobserved in kidney cancer pool. Expression was also detected in VP1,prostate cancer xenograft pool, prostate cancer pool and colon cancerpool. Low expression was observed in VP2, lung cancer pool, ovary cancerpool, breast cancer pool, metastasis pool, pancreas cancer pool, and inthe 2 different prostate cancer metastasis to lymph node.

Example 2 Isolation of Full Length 161P2F10B Encoding cDNA

To isolate genes that are involved in kidney cancer, an experiment wasconducted using kidney cancer patient specimens. The gene 161P2F10B wasderived from a subtraction consisting of kidney cancer specimens, minusnormal kidney mixed with a cocktail of 9 normal tissues: stomach,skeletal muscle, lung, brain, liver, kidney, pancreas, small intestineand heart. The SSH DNA sequence (FIG. 1) was designated 161P2F10B. cDNAclone 161P2F10B was cloned from kidney cancer specimens (FIG. 2 and FIG.3). 161P2F10B showed homology to the gene ENPP3. The amino acidalignment of 161P2F10B with ENPP3 is shown in FIG. 4 (also, see, e.g.,Buhring, et al., Blood 97:3303-3305 (2001)).

Example 3 Chromosomal Mapping of 161P2F10B

Chromosomal localization can implicate genes in disease pathogenesis.Several chromosome mapping approaches are available includingfluorescent in situ hybridization (FISH), human/hamster radiation hybrid(RH) panels (Walter et al., 1994; Nature Genetics 7:22; ResearchGenetics, Huntsville Ala.), human-rodent somatic cell hybrid panels suchas is available from the Coriell Institute (Camden, N.J.), and genomicviewers utilizing BLAST homologies to sequenced and mapped genomicclones (NCBI, Bethesda, Md.). 161P2F10B maps to chromosome 6q22, using161P2F10B sequence and the NCBI BLAST tool located on the World Wide Website of the National Institutes of Health.

Example 4 Expression Analysis of 161P2F10B

To compare expression of 161P2F10B in normal versus patient cancertissues, RT-PCR experiment was performed using normal and patient cancertissues (FIG. 14). First strand cDNA was generated from normal stomach,normal brain, normal heart, normal liver, normal skeletal muscle, normaltestis, normal prostate, normal bladder, normal kidney, normal colon,normal lung, normal pancreas, and a pool of cancer specimens fromprostate cancer patients, bladder cancer patients, kidney cancerpatients, colon cancer patients, lung cancer patients, pancreas cancerpatients, a pool of prostate cancer xenografts (LAPC-4AD, LAPC-4AI,LAPC-9AD and LAPC-9AI), and a pool of 2 patient prostate metastasis tolymph node. Normalization was performed by PCR using primers to actin.Semi-quantitative PCR, using primers to 161P2F10B, was performed at 26and 30 cycles of amplification. Samples were run on an agarose gel, andPCR products were quantitated using the Alphalmager software. Resultsshow strong expression in prostate cancer, bladder cancer, kidneycancer, colon cancer, lung cancer, pancreas cancer, bone cancer,lymphoma cancer, uterus cancer, compared to all normal tissues tested.Strong expression was also detected in the xenograft pool as well as theprostate cancer metastasis to lymph node specimens.

FIG. 15 & Table LIX shows expression of 161P2F10B in a panel of kidneycancer clear cell carcinoma (A), kidney cancer papillary carcinoma (B),and in uterus patient cancer specimens (C). First strand cDNA wasprepared from the patient specimens. Normalization was performed by PCRusing primers to actin. Semi-quantitative PCR, using primers to161P2F10B, was performed at 26 and 30 cycles of amplification. Sampleswere run on an agarose gel, and PCR products were quantitated using theAlphalmager software. Expression was recorded as absent, low, medium orstrong. Results show expression of 161P2F10B in 94.7% of clear cellrenal carcinoma, 62.5% of papillary renal cell carcinoma, and in 61.5%of uterus cancer.

The restricted expression of 161P2F10B in normal tissues and theupregulation detected in kidney cancer, in kidney cancer metastasis, aswell as in prostate, bladder, colon, lung, pancreas, bone, lymphoma,uterus, breast, and ovary cancers, suggest that 161P2F10B is a potentialtherapeutic target and a diagnostic marker for human cancers.

Example 5 Transcript Variants of 161P2F10B

Transcript variants are variants of mature mRNA from the same gene,which arise by alternative transcription or alternative splicing.Alternative transcripts are transcripts from the same gene but starttranscription at different points. Splice variants are mRNA variantsspliced differently from the same transcript. In eukaryotes, when amulti-exon gene is transcribed from genomic DNA, the initial RNA isspliced to produce functional mRNA, which has only exons and is used fortranslation into an amino acid sequence. Accordingly, a given gene canhave zero to many alternative transcripts and each transcript can havezero to many splice variants. Each transcript variant has a unique exonmakeup, and can have different coding and/or non-coding (5′ or 3′ end)portions, from the original transcript. Transcript variants can code forsimilar or different proteins with the same or a similar function or canencode proteins with different functions, and can be expressed in thesame tissue at the same time, or in different tissues at the same time,or in the same tissue at different times, or in different tissues atdifferent times. Proteins encoded by transcript variants can havesimilar or different cellular or extracellular localizations, e.g.,secreted versus intracellular.

Transcript variants are identified by a variety of art-accepted methods.For example, alternative transcripts and splice variants are identifiedby full-length cloning experiment, or by use of full-length transcriptand EST sequences. First, all human ESTs were grouped into clusterswhich show direct or indirect identity with each other. Second, ESTs inthe same cluster were further grouped into sub-clusters and assembledinto a consensus sequence. The original gene sequence is compared to theconsensus sequence(s) or other full-length sequences. Each consensussequence is a potential splice variant for that gene. Even when avariant is identified that is not a full-length clone, that portion ofthe variant is very useful for antigen generation and for furthercloning of the full-length splice variant, using techniques known in theart.

Moreover, computer programs are available in the art that identifytranscript variants based on genomic sequences. Genomic-based transcriptvariant identification programs include FgenesH (A. Salamov and V.Solovyev, “Ab initio gene finding in Drosophila genomic DNA,” GenomeResearch. 2000 April; 10(4):516-22); Grail (URLcompbio.ornl.gov/Grail-bin/EmptyGrailForm) and GenScan (URLgenes.mit.edu/GENSCAN.html). For a general discussion of splice variantidentification protocols see., e.g., Southan, C., A genomic perspectiveon human proteases, FEBS Lett. 2001 Jun. 8; 498(2-3):214-8; de Souza, S.J., et al., Identification of human chromosome 22 transcribed sequenceswith ORF expressed sequence tags, Proc. Natl Acad Sci USA. 2000 Nov. 7;97(23):12690-3.

To further confirm the parameters of a transcript variant, a variety oftechniques are available in the art, such as full-length cloning,proteomic validation, PCR-based validation, and 5′ RACE validation, etc.(see e.g., Proteomic Validation: Brennan, S. O., et al., Albumin bankspeninsula: a new termination variant characterized by electrospray massspectrometry, Biochem Biophys Acta. 1999 Aug. 17; 1433(1-2):321-6;Ferranti P, et al., Differential splicing of pre-messenger RNA producesmultiple forms of mature caprine alpha(s1)-casein, Eur J Biochem. 1997Oct. 1; 249(1):1-7. For PCR-based Validation: Wellmann S, et al.,Specific reverse transcription-PCR quantification of vascularendothelial growth factor (VEGF) splice variants by LightCyclertechnology, Clin Chem. 2001 April; 47(4):654-60; Jia, H. P., et al.,Discovery of new human beta-defensins using a genomics-based approach,Gene. 2001 Jan. 24; 263(1-2):211-8. For PCR-based and 5′ RACEValidation: Brigle, K. E., et al., Organization of the murine reducedfolate carrier gene and identification of variant splice forms, BiochemBiophys Acta. 1997 Aug. 7; 1353(2): 191-8).

It is known in the art that genomic regions are modulated in cancers.When the genomic region to which a gene maps is modulated in aparticular cancer, the alternative transcripts or splice variants of thegene are modulated as well. Disclosed herein is that 161P2F10B has aparticular expression profile related to cancer. Alternative transcriptsand splice variants of 161P2F10B may also be involved in cancers in thesame or different tissues, thus serving as tumor-associatedmarkers/antigens.

Using the full-length gene and EST sequences, two transcript variantswere identified, designated as 161P2F10B v.6 and v.7. Compared with161P2F10B v.1, transcript variant 161P2F10B v.6 has extra 40 bases tothe 5′ starting site of variant 161P2F10B v.1 transcript and has adifferent 3′ end portion, which is on the same chromosome as other exonsin the current version of human genome. Variant 161P2F10B v.7 inserted130 bases in between positions 121 and 122 of variant 161P2F10B v.1.Theoretically, each different combination of exons in spatial order,e.g. exons 2 and 3, is a potential splice variant. Due to the incorrectassembly of the chromosome region in the current version of humangenome, the transcript structure cannot be derived computationally.

Tables LI through LVIII are set forth on a variant by variant bases.Tables LI and LV show the nucleotide sequence of the transcript variant.Tables LII and LVI show the alignment of the transcript variant withnucleic acid sequence of 161P2F10B v.1. Tables LIII and LVII lay outamino acid translation of the transcript variant for the identifiedreading frame orientation. Tables LIV and LVIII display alignments ofthe amino acid sequence encoded by the splice variant with that of161P2F10B v.1.

Example 6 Single Nucleotide Polymorphisms of 161P2F10B

A Single Nucleotide Polymorphism (SNP) is a single base pair variationin a nucleotide sequence at a specific location. At any given point ofthe genome, there are four possible nucleotide base pairs: A/T, C/G, G/Cand T/A. Genotype refers to the specific base pair sequence of one ormore locations in the genome of an individual. Haplotype refers to thebase pair sequence of more than one location on the same DNA molecule(or the same chromosome in higher organisms), often in the context ofone gene or in the context of several tightly linked genes. SNPs thatoccur on a cDNA are called cSNPs. These cSNPs may change amino acids ofthe protein encoded by the gene and thus change the functions of theprotein. Some SNPs cause inherited diseases; others contribute toquantitative variations in phenotype and reactions to environmentalfactors including diet and drugs among individuals. Therefore, SNPsand/or combinations of alleles (called haplotypes) have manyapplications, including diagnosis of inherited diseases, determinationof drug reactions and dosage, identification of genes responsible fordiseases, and analysis of the genetic relationship between individuals(P. Nowotny, J. M. Kwon and A. M. Goate, “ SNP analysis to dissect humantraits,” Curr. Opin. Neurobiol. 2001 October; 11(5):637-641; M.Pirmohamed and B. K. Park, “Genetic susceptibility to adverse drugreactions,” Trends Pharmacol. Sci. 2001 June; 22(6):298-305; J. H.Riley, C. J. Allan, E. Lai and A. Roses, “The use of single nucleotidepolymorphisms in the isolation of common disease genes,”Pharmacogenomics. 2000 February; 1(1):39-47; R. Judson, J. C. Stephensand A. Windemuth, “The predictive power of haplotypes in clinicalresponse,” Pharmacogenomics. 2000 February; 1(1):15-26).

SNPs are identified by a variety of art-accepted methods (P. Bean, “Thepromising voyage of SNP target discovery,” Am. Clin. Lab. 2001October-November; 20(9):18-20; K. M. Weiss, “In search of humanvariation,” Genome Res. 1998 July; 8(7):691-697; M. M. She, “Enablinglarge-scale pharmacogenetic studies by high-throughput mutationdetection and genotyping technologies,” Clin. Chem. 2001 February;47(2):164-172). For example, SNPs are identified by sequencing DNAfragments that show polymorphism by gel-based methods such asrestriction fragment length polymorphism (RFLP) and denaturing gradientgel electrophoresis (DGGE). They can also be discovered by directsequencing of DNA samples pooled from different individuals or bycomparing sequences from different DNA samples. With the rapidaccumulation of sequence data in public and private databases, one candiscover SNPs by comparing sequences using computer programs (Z. Gu, L.Hillier and P. Y. Kwok, “Single nucleotide polymorphism hunting incyberspace,” Hum. Mutat. 1998; 12(4):221-225). SNPs can be verified andgenotype or haplotype of an individual can be determined by a variety ofmethods including direct sequencing and high throughput microarrays (P.Y. Kwok, “Methods for genotyping single nucleotide polymorphisms,” Annu.Rev. Genomics Hum. Genet. 2001; 2:235-258; M. Kokoris, K. Dix, K.Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A. Duesterhoeft,“High-throughput SNP genotyping with the Masscode system,” Mol. Diagn.2000 December; 5(4):329-340).

Using the methods described above, four SNPs were identified in theoriginal transcript, 161P2F10B v.1, at positions 408 (A/G), 2502 (A/G),2663 (A/C) and 3233 (A/C). The transcripts or proteins with alternativealleles were designated as variants 161P2F10B v.2, v.3, v.4, and v.5,respectively. FIG. 10 shows the schematic alignment of the SNP variants.FIG. 11 shows the schematic alignment of protein variants, correspondingto nucleotide variants. Nucleotide variants that code for the same aminoacid sequence as variant 1 are not shown in FIG. 11. These alleles ofthe SNPs, though shown separately here, can occur in differentcombinations (haplotypes) and in any one of the transcript variants(such as 161P2F10B v.7) that contains the sequence context of the SNPs.

Example 7 Production of Recombinant 161P2F10B in Prokaryotic Systems

To express recombinant 161P2F10B in prokaryotic cells, the full orpartial length 161P2F10B cDNA sequences can be cloned into any one of avariety of expression vectors known in the art. One or more of thefollowing regions of 161P2F10B are expressed in these contructs, aminoacids 1 to 875; or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acidsfrom 161P2F10B, variants, or analogs thereof.

A. In Vitro Transcription and Translation Constructs:

pCRII: To generate 161P2F10B sense and anti-sense RNA probes for RNA insitu investigations, pCRII constructs (Invitrogen, Carlsbad Calif.) aregenerated encoding either all or fragments of the 161P2F10B cDNA. ThepCRII vector has Sp6 and T7 promoters flanking the insert to drive thetranscription of 161P2F10B RNA for use as probes in RNA in situhybridization experiments. These probes are used to analyze the cell andtissue expression of 161P2F10B at the RNA level. Transcribed 161P2F10BRNA representing the cDNA amino acid coding region of the 161P2F10B geneis used in in vitro translation systems such as the TnTTM CoupledReticulolysate System (Promega, Corp., Madison, Wis.) to synthesize161P2F10B protein.

B. Bacterial Constructs:

pGEX Constructs: To generate recombinant 161P2F10B proteins in bacteriathat are fused to the Glutathione S-transferase (GST) protein, all orparts of the 161P2F10B cDNA protein coding sequence are fused to the GSTgene by cloning into pGEX-6P-1 or any other GST-fusion vector of thepGEX family (Amersham Pharmacia Biotech, Piscataway, N.J.). Theseconstructs allow controlled expression of recombinant 161P2F10B proteinsequences with GST fused at the amino-terminus and a six histidineepitope (6× His) at the carboxyl-terminus The GST and 6× His tags permitpurification of the recombinant fusion protein from induced bacteriawith the appropriate affinity matrix and allow recognition of the fusionprotein with anti-GST and anti-His antibodies. The 6× His tag isgenerated by adding 6 histidine codons to the cloning primer at the 3′end, e.g., of the open reading frame (ORF). A proteolytic cleavage site,such as the PreScissionTM recognition site in pGEX-6P-1, may be employedsuch that it permits cleavage of the GST tag from 161P2F10B-relatedprotein. The ampicillin resistance gene and pBR322 origin permitsselection and maintenance of the pGEX plasmids in E. coli.

pMAL Constructs: To generate, in bacteria, recombinant 161P2F10Bproteins that are fused to maltose-binding protein (MBP), all or partsof the 161P2F10B cDNA protein coding sequence are fused to the MBP geneby cloning into the pMAL-c2X and pMAL-p2X vectors (New England Biolabs,Beverly, Mass.). These constructs allow controlled expression ofrecombinant 161P2F10B protein sequences with MBP fused at theamino-terminus and a 6× His epitope tag at the carboxyl-terminus The MBPand 6× His tags permit purification of the recombinant protein frominduced bacteria with the appropriate affinity matrix and allowrecognition of the fusion protein with anti-MBP and anti-His antibodies.The 6× His epitope tag is generated by adding 6 histidine codons to the3′ cloning primer. A Factor Xa recognition site permits cleavage of thepMAL tag from 161P2F10B. The pMAL-c2X and pMAL-p2X vectors are optimizedto express the recombinant protein in the cytoplasm or periplasmrespectively. Periplasm expression enhances folding of proteins withdisulfide bonds.

pET Constructs: To express 161P2F10B in bacterial cells, all or parts ofthe 161P2F10B cDNA protein coding sequence are cloned into the pETfamily of vectors (Novagen, Madison, Wis.). These vectors allow tightlycontrolled expression of recombinant 161P2F10B protein in bacteria withand without fusion to proteins that enhance solubility, such as NusA andthioredoxin (Trx), and epitope tags, such as 6× His and S-Tag™ that aidpurification and detection of the recombinant protein. For example,constructs are made utilizing pET NusA fusion system 43.1 such thatregions of the 161P2F10B protein are expressed as amino-terminal fusionsto NusA.

C. Yeast Constructs:

pESC Constructs: To express 161P2F10B in the yeast species Saccharomycescerevisiae for generation of recombinant protein and functional studies,all or parts of the 161P2F10B cDNA protein coding sequence are clonedinto the pESC family of vectors each of which contain 1 of 4 selectablemarkers, HIS3, TRP1, LEU2, and URA3 (Stratagene, La Jolla, Calif.).These vectors allow controlled expression from the same plasmid of up to2 different genes or cloned sequences containing either F1agTM or Mycepitope tags in the same yeast cell. This system is useful to confirmprotein-protein interactions of 161P2F10B. In addition, expression inyeast yields similar post-translational modifications, such asglycosylations and phosphorylations, that are found when expressed ineukaryotic cells.

pESP Constructs: To express 161P2F10B in the yeast species Saccharomycespombe, all or parts of the 161P2F10B cDNA protein coding sequence arecloned into the pESP family of vectors. These vectors allow controlledhigh level of expression of a 161P2F10B protein sequence that is fusedat either the amino terminus or at the carboxyl terminus to GST whichaids purification of the recombinant protein. A FlagTM epitope tagallows detection of the recombinant protein with anti-FlagTM antibody.

Example 8 Production of Recombinant 161P2F10B in Higher EukaryoticSystems

A. Mammalian Constructs:

To express recombinant 161P2F10B in eukaryotic cells, the full orpartial length 161P2F10B cDNA sequences can be cloned into any one of avariety of expression vectors known in the art. One or more of thefollowing regions of 161P2F10B are expressed in these constructs, aminoacids 1 to 875; or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acidsfrom 161P2F10B, variants, or analogs thereof.

The constructs were transfected into any one of a wide variety ofmammalian cells such as 293T cells or kidney cancer cell lines.Transfected 293T cell lysates were probed with the anti-161P2F10Bpolyclonal serum and monoclonal antibodies, described herein.

pcDNA3.1/MycHis Constructs: To express 161P2F10B in mammalian cells, the161P2F10B ORF, or portions thereof, of 161P2F10B with a consensus Kozaktranslation initiation site were cloned into pcDNA3.1/MycHis Version A(Invitrogen, Carlsbad, Calif.). Protein expression is driven from thecytomegalovirus (CMV) promoter. The recombinant proteins have the mycepitope and 6× His epitope fused to the carboxyl-terminus ThepcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH)polyadenylation signal and transcription termination sequence to enhancemRNA stability, along with the SV40 origin for episomal replication andsimple vector rescue in cell lines expressing the large T antigen. TheNeomycin resistance gene can be used, as it allows for selection ofmammalian cells expressing the protein and the ampicillin resistancegene and ColE1 origin permits selection and maintenance of the plasmidin E. coli.

The pcDNA3.1/mycHis encoding 161P2F10B was transfected in 293T cells.Cells were harvested 24 hours later and analyzed showing cell surfaceexpression of 161P2F10B driven from the pcDNA3.1/mycHis vector (FIG.29).

pTag5: The 161P2F10B ORF, or portions thereof, of 161P2F10B were clonedinto pTag-5. This vector is similar to pAPtag but without the alkalinephosphatase fusion. This construct generates 161P2F10B protein with anamino-terminal IgGκ signal sequence and myc and 6× His epitope tags atthe carboxyl-terminus that facilitate detection and affinitypurification. The resulting recombinant 161P2F10B protein was optimizedfor secretion into the media of transfected mammalian cells, and wasused as immunogen or ligand to identify proteins such as ligands orreceptors that interact with the 161P2F10B proteins. Protein expressionis driven from the CMV promoter. The Zeocin resistance gene present inthe vector allows for selection of mammalian cells expressing theprotein, and the ampicillin resistance gene permits selection of theplasmid in E. coli. FIGS. 31 and 32 show expression and enzymaticactivity of the soluble pTag5 expressing 161P2F10B.

PsecFc: The 161P2F10B ORF, or portions thereof, of 161P2F10B were clonedinto psecFc. The psecFc vector was assembled by cloning the humanimmunoglobulin G1 (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2(Invitrogen, California). This construct generates an IgG1 Fc fusion atthe amino-terminus of the 161P2F10B proteins. 161P2F10B fusionsutilizing the murine IgG1 Fc region was also generated and expressed.The resulting recombinant 161P2F10B proteins are optimized for secretioninto the media of transfected mammalian cells, and can be used asimmunogens or to identify proteins such as ligands or receptors thatinteract with the 161P2F10B protein. Protein expression is driven fromthe CMV promoter. The hygromycin resistance gene present in the vectorallows for selection of mammalian cells that express the recombinantprotein, and the ampicillin resistance gene permits selection of theplasmid in E. coli.

pSRα Constructs: To generate mammalian cell lines that express 161P2F10Bconstitutively, 161P2F10B ORF, or portions thereof, of 161P2F10B arecloned into pSRα constructs. Amphotropic and ecotropic retroviruses weregenerated by transfection of pSRα constructs into the 293T-10A1packaging line or co-transfection of pSRα and a helper plasmid(containing deleted packaging sequences) into the 293 cells. Theretrovirus is used to infect a variety of mammalian cell lines,resulting in the integration of the cloned gene, 161P2F10B, into thehost cell-lines. Protein expression is driven from a long terminalrepeat (LTR). The Neomycin resistance gene present in the vector allowsfor selection of mammalian cells that express the protein, and theampicillin resistance gene and ColE1 origin permit selection andmaintenance of the plasmid in E. coli. The retroviral vectors werethereafter used for infection and generation of various cell linesusing, for example, NIH 3T3, 293 Rat-1 cells or kidney cancer cell linessuch as Caki and 769 cells. FIGS. 16 and 30 show cell surface expressionof 161P2F10B driven from the pSRa construct in Caki and NIH3T3 cellsrespectively.

Additional pSRa constructs were generated encoding 3 different mutantsof 161P2F10B. The first mutant is D80E, converted the D amino acidresidue of the RGD domain at position 80 into E. The other mutants aremutants of the active site of 161P2F10B, converting the T205 amino acidresidue at position 205 into either A (T205A), or S (T205S). The 3mutant pSRa constructs were transfected into a variety of mammalian celllines such as 293T cells and CaKi kidney cancer cells. Expression wasconfirmed using anti-161P2F10B monoclonal antibody and phosphodiesteraseenzyme activity was tested (FIG. 30).

pcDNA4/HisMax Constructs: To express 161P2F10B in mammalian cells, the161P2F10B ORF, or portions thereof, of 161P2F10B are cloned intopcDNA4/HisMax Version A (Invitrogen, Carlsbad, Calif.). Proteinexpression is driven from the cytomegalovirus (CMV) promoter and theSP16 translational enhancer. The recombinant protein has XpressTM andsix histidine (6× His) epitopes fused to the amino-terminus. ThepcDNA4/HisMax vector also contains the bovine growth hormone (BGH)polyadenylation signal and transcription termination sequence to enhancemRNA stability along with the SV40 origin for episomal replication andsimple vector rescue in cell lines expressing the large T antigen. TheZeocin resistance gene allows for selection of mammalian cellsexpressing the protein and the ampicillin resistance gene and ColE1origin permits selection and maintenance of the plasmid in E. coli.

pcDNA3.1/CT-GFP-TOPO Construct: To express 161P2F10B in mammalian cellsand to allow detection of the recombinant proteins using fluorescence,the 161P2F10B ORF, or portions thereof, of 161P2F10B with a consensusKozak translation initiation site are cloned into pcDNA3.1/CT-GFP-TOPO(Invitrogen, CA). Protein expression is driven from the cytomegalovirus(CMV) promoter. The recombinant proteins have the Green FluorescentProtein (GFP) fused to the carboxyl-terminus facilitating non-invasive,in vivo detection and cell biology studies. The pcDNA3.1/CT-GFP-TOPOvector also contains the bovine growth hormone (BGH) polyadenylationsignal and transcription termination sequence to enhance mRNA stabilityalong with the SV40 origin for episomal replication and simple vectorrescue in cell lines expressing the large T antigen. The Neomycinresistance gene allows for selection of mammalian cells that express theprotein, and the ampicillin resistance gene and ColE1 origin permitsselection and maintenance of the plasmid in E. coli. Additionalconstructs with an amino-terminal GFP fusion are made inpcDNA3.1/NT-GFP-TOPO spanning the entire length of the 161P2F10Bproteins.

PAPtag: The 161P2F10B ORF, or portions thereof, of 161P2F10B are clonedinto pAPtag-5 (GenHunter Corp. Nashville, Tenn.). This constructgenerates an alkaline phosphatase fusion at the carboxyl-terminus of the161P2F10B proteins while fusing the IgGκ signal sequence to theamino-terminus Constructs are also generated in which alkalinephosphatase with an amino-terminal IgGκ signal sequence is fused to theamino-terminus of 161P2F10B proteins. The resulting recombinant161P2F10B proteins are optimized for secretion into the media oftransfected mammalian cells and can be used to identify proteins such asligands or receptors that interact with the 161P2F10B proteins. Proteinexpression is driven from the CMV promoter and the recombinant proteinsalso contain myc and 6× His epitopes fused at the carboxyl-terminus thatfacilitates detection and purification. The Zeocin resistance genepresent in the vector allows for selection of mammalian cells expressingthe recombinant protein and the ampicillin resistance gene permitsselection of the plasmid in E. coli.

Additional Viral Vectors: Additional constructs are made forviral-mediated delivery and expression of 161P2F10B. High virus titerleading to high-level expression of 161P2F10B is achieved in viraldelivery systems such as adenoviral vectors and herpes amplicon vectors.The 161P2F10B coding sequences or fragments thereof are amplified by PCRand subcloned into the AdEasy shuttle vector (Stratagene). Recombinationand virus packaging are performed according to the manufacturer'sinstructions to generate adenoviral vectors. Alternatively, 161P2F10Bcoding sequences or fragments thereof are cloned into the HSV-1 vector(Imgenex) to generate herpes viral vectors. The viral vectors arethereafter used for infection of various cell lines such as PC3, NIH3T3, 293 or rat-1 cells.

Regulated Expression Systems: To control expression of 161P2F10B inmammalian cells, coding sequences of 161P2F10B, or portions thereof, arecloned into regulated mammalian expression systems such as the T-RexSystem (Invitrogen), the GeneSwitch System (Invitrogen) and thetightly-regulated Ecdysone System (Sratagene). These systems allow thestudy of the temporal and concentration dependent effects of recombinant161P2F10B. These vectors are thereafter used to control expression of161P2F10B in various cell lines such as PC3, NIH 3T3, 293 or rat-1cells.

B. Baculovirus Expression Systems

To generate recombinant 161P2F10B proteins in a Baculovirus expressionsystem, 161P2F10B ORF, or portions thereof, are cloned into theBaculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides aHis-tag at the N-terminus Specifically, pBlueBac-161P2F10B isco-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9(Spodoptera frugiperda) insect cells to generate recombinant Baculovirus(see Invitrogen instruction manual for details). Baculovirus is thencollected from cell supernatant and purified by plaque assay.

Recombinant 161P2F10B protein is then generated by infection of HighFiveinsect cells (Invitrogen) with purified Baculovirus. Recombinant161P2F10B protein can be detected using anti-161P2F10B or anti-His-tagantibody. 161P2F10B protein can be purified and used in variouscell-based assays or as immunogen to generate polyclonal and monoclonalantibodies specific for 161P2F10B.

Example 9 Antigenicity Profiles and Secondary Structure

FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9 depict graphically five aminoacid profiles of the 161P2F10B amino acid sequence, each assessmentavailable by accessing the ProtScale website located on the World WideWeb on the ExPasy molecular biology server.

These profiles: FIG. 5, Hydrophilicity, (Hopp T. P., Woods K. R., 1981.Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); FIG. 6, Hydropathicity,(Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132); FIG. 7,Percentage Accessible Residues (Janin J., 1979 Nature 277:491-492); FIG.8, Average Flexibility, (Bhaskaran R., and Ponnuswamy P. K., 1988. Int.J. Pept. Protein Res. 32:242-255); FIG. 9, Beta-turn (Deleage, G., RouxB. 1987 Protein Engineering 1:289-294); and optionally others availablein the art, such as on the ProtScale website, were used to identifyantigenic regions of the 161P2F10B protein. Each of the above amino acidprofiles of 161P2F10B were generated using the following ProtScaleparameters for analysis: 1) A window size of 9; 2) 100% weight of thewindow edges compared to the window center; and, 3) amino acid profilevalues normalized to lie between 0 and 1.

Hydrophilicity (FIG. 5), Hydropathicity (FIG. 6) and PercentageAccessible Residues (FIG. 7) profiles were used to determine stretchesof hydrophilic amino acids (i.e., values greater than 0.5 on theHydrophilicity and Percentage Accessible Residues profile, and valuesless than 0.5 on the Hydropathicity profile). Such regions are likely tobe exposed to the aqueous environment, be present on the surface of theprotein, and thus available for immune recognition, such as byantibodies.

Average Flexibility (FIG. 8) and Beta-turn (FIG. 9) profiles determinestretches of amino acids (i.e., values greater than 0.5 on the Beta-turnprofile and the Average Flexibility profile) that are not constrained insecondary structures such as beta sheets and alpha helices. Such regionsare also more likely to be exposed on the protein and thus accessible toimmune recognition, such as by antibodies.

Antigenic sequences of the 161P2F10B protein indicated, e.g., by theprofiles set forth in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and/or FIG. 9 areused to prepare immunogens, either peptides or nucleic acids that encodethem, to generate therapeutic and diagnostic anti-161P2F10B antibodies.The immunogen can be any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more than 50contiguous amino acids, or the corresponding nucleic acids that encodethem, from the 161P2F10B protein. In particular, peptide immunogens ofthe invention can comprise, a peptide region of at least 5 amino acidsof FIG. 2 in any whole number increment up to 875 that includes an aminoacid position having a value greater than 0.5 in the Hydrophilicityprofile of FIG. 5; a peptide region of at least 5 amino acids of FIG. 2in any whole number increment up to 875 that includes an amino acidposition having a value less than 0.5 in the Hydropathicity profile ofFIG. 6; a peptide region of at least 5 amino acids of FIG. 2 in anywhole number increment up to 875 that includes an amino acid positionhaving a value greater than 0.5 in the Percent Accessible Residuesprofile of FIG. 7; a peptide region of at least 5 amino acids of FIG. 2in any whole number increment up to 875 that includes an amino acidposition having a value greater than 0.5 in the Average Flexibilityprofile on FIG. 8; and, a peptide region of at least 5 amino acids ofFIG. 2 in any whole number increment up to 875 that includes an aminoacid position having a value greater than 0.5 in the Beta-turn profileof FIG. 9. Peptide immunogens of the invention can also comprise nucleicacids that encode any of the forgoing.

All immunogens of the invention, peptide or nucleic acid, can beembodied in human unit dose form, or comprised by a composition thatincludes a pharmaceutical excipient compatible with human physiology.

The secondary structure of 161P2F10B, namely the predicted presence andlocation of alpha helices, extended strands, and random coils, ispredicted from the primary amino acid sequence using theHNN—Hierarchical Neural Network method accessed from the ExPasymolecular biology server. The analysis indicates that 161P2F10B iscomposed 31.31% alpha helix, 11.31% extended strand, and 57.37% randomcoil (FIG. 19A).

Analysis for the potential presence of transmembrane domains in161P2F10B was carried out using a variety of transmembrane predictionalgorithms accessed from the ExPasy molecular biology server. Theprograms predict the presence of 1 transmembrane domain in 161P2F10B,consistent with that of a Type II cell surface protein. Showngraphically in FIG. 19 are the results of analysis using the TMpred(FIG. 19B) and TMHMM (FIG. 19C) prediction programs depicting thelocation of the transmembrane domain.

Example 10 Generation of 161P2F10B Polyclonal Antibodies

Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Inaddition to immunizing with the full length 161P2F10B protein, computeralgorithms are employed in design of immunogens that, based on aminoacid sequence analysis contain characteristics of being antigenic andavailable for recognition by the immune system of the immunized host(see the Example entitled “Antigenicity Profiles”). Such regions wouldbe predicted to be hydrophilic, flexible, in beta-turn conformations,and be exposed on the surface of the protein (see, e.g., FIG. 5, FIG. 6,FIG. 7, FIG. 8, or FIG. 9 for amino acid profiles that indicate suchregions of 161P2F10B).

For example, 161P2F10B recombinant bacterial fusion proteins or peptidescontaining hydrophilic, flexible, beta-turn regions of the 161P2F10B, inwhich numerous regions are found in the predicted extracellular domaincoded by amino acids 45-870, are used as antigens to generate polyclonalantibodies in New Zealand White rabbits. For example, such regionsinclude, but are not limited to, amino acids 43-93, 100-134,211-246,467-492, 500-517, and amino acids 810-870. In addition,recombinant proteins are made that encode the whole extracellulardomain, amino acids 45-870, or halves of the domain, such as amino acids45-450 and amino acids 451-870. Antigens are also created encoding theSomatomedin-B-like domain (amino acids 53-133), the catalytic domain(amino acids 158-538), and the nuclease like domain (amino acids609-875) of 161P2F10B (Bollen et. al., 2000. Crit. Rev. Biochem. Mol.Biol., 35: 393-432), in order to generate antibodies specific to theseregions. Ideally antibodies are raised to non-conserved regions of thesedomains such that they do not crossreact with other homologousnucleotide pyrophosphatases/phosphodiesterases. It is useful toconjugate the immunizing agent to a protein known to be immunogenic inthe mammal being immunized. Examples of such immunogenic proteinsinclude, but are not limited to, keyhole limpet hemocyanin (KLH), serumalbumin, bovine thyroglobulin, and soybean trypsin inhibitor. In oneembodiment, a peptide encoding amino acids 500-517 of 161P2F10B isconjugated to KLH and used to immunize the rabbit. Alternatively theimmunizing agent may include all or portions of the 161P2F10B protein,analogs or fusion proteins thereof. For example, the 161P2F10B aminoacid sequence can be fused using recombinant DNA techniques to any oneof a variety of fusion protein partners that are well known in the art,such as glutathione-S-transferase (GST) and HIS tagged fusion proteins.Such fusion proteins are purified from induced bacteria using theappropriate affinity matrix.

In one embodiment, a GST-fusion protein encoding amino acids 45-875 isproduced and purified and used as immunogen. Other recombinant bacterialfusion proteins that may be employed include maltose binding protein,LacZ, thioredoxin, NusA, or an immunoglobulin constant region (see thesection entitled “Production of 161P2F10B in Prokaryotic Systems” andCurrent Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M.Ausubul et al. eds., 1995; Linsley, P. S., Brady, W., Urnes, M.,Grosmaire, L., Damle, N., and Ledbetter, L. (1991) J. Exp. Med. 174,561-566).

In addition to bacterial derived fusion proteins, mammalian expressedprotein antigens are also used. These antigens are expressed frommammalian expression vectors such as the TagS and Fc-fusion vectors (seethe section entitled “Production of Recombinant 161P2F10B in EukaryoticSystems”), and retain post-translational modifications such asglycosylations found in native protein. In one embodiment, amino acids45-875 is cloned into the TagS mammalian secretion vector. Therecombinant protein is purified by metal chelate chromatography fromtissue culture supernatants of 293T cells stably expressing therecombinant vector. The purified TagS 161P2F10B protein is then used asimmunogen.

During the immunization protocol, it is useful to mix or emulsify theantigen in adjuvants that enhance the immune response of the hostanimal. Examples of adjuvants include, but are not limited to, completeFreund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate).

In a typical protocol, rabbits are initially immunized subcutaneouslywith up to 200 μg, typically 100-200 μg, of fusion protein or peptideconjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits arethen injected subcutaneously every two weeks with up to 200 μg,typically 100-200 μg, of the immunogen in incomplete Freund's adjuvant(IFA). Test bleeds are taken approximately 7-10 days following eachimmunization and used to monitor the titer of the antiserum by ELISA.

To test reactivity and specificity of immune serum, such as the rabbitserum derived from immunization with TagS 161P2F10B encoding amino acids58-538, the full-length 161P2F10B cDNA is cloned into pCDNA 3.1 myc-hisexpression vector (Invitrogen, see the Example entitled “Production ofRecombinant 161P2F10B in Eukaryotic Systems”). After transfection of theconstructs into 293T cells, cell lysates are probed with theanti-161P2F10B serum and with anti-His antibody (Santa CruzBiotechnologies, Santa Cruz, Calif.) to determine specific reactivity todenatured 161P2F10B protein using the Western blot techniqueImmunoprecipitation and flow cytometric analyses of 293T and otherrecombinant 161P2F10B-expressing cells determine recognition of nativeprotein by the antiserum. In addition, Western blot,immunoprecipitation, fluorescent microscopy, and flow cytometrictechniques using cells that endogenously express 161P2F10B are carriedout to test specificity.

The anti-serum from the TagS 161P2F10B immunized rabbit is affinitypurified by passage over a column composed of the TagS antigencovalently coupled to Affigel matrix (BioRad, Hercules, Calif.). Theserum is then further purified by protein G affinity chromatography toisolate the IgG fraction. Serum from rabbits immunized with fusionproteins, such as GST and MBP fusion proteins, are purified by depletionof antibodies reactive to the fusion partner sequence by passage over anaffinity column containing the fusion partner either alone or in thecontext of an irrelevant fusion protein. Sera from other His-taggedantigens and peptide immunized rabbits as well as fusion partnerdepleted sera are affinity purified by passage over a column matrixcomposed of the original protein immunogen or free peptide.

Example 11 Generation of 161P2F10B Monoclonal Antibodies (mAbs)

The use of agents to identify the presence of 161P2F10Bin biopsyspecimens or to neutralize the effect of 161P2F10B has a beneficialeffect in diagnosis, prognoosis, prophylaxis and/or therapy. Oneparticularly useful class of anti 161P2F10B agents is antibodies, inparticular monoclonal antibodies (Mabs) to 161P2F10B. Anti 161P2F10BAbs, such as Mabs, are generated that react with the epitopes of the161P2F10B protein such that they either indicate it's presence, disruptor modulate it's biological function (for example those that woulddisrupt the interaction with ligands or proteins that mediate or areinvolved in it's biological activity) or are able to carry a toxin tothe cell which is expressing 161P2F10B.

The term anti 161P2F10B antibody as used herein is to be understood tocover antibodies to any epitope of the 161P2F10B gene product.Diagnostic Mabs, e.g. those used for imaging or immunocytochemistry,comprise those that specifically bind epitopes of 161P2F10B protein andthus demonstrate its presence. Therapeutic Mabs include those that areuseful for diagnosis but also comprise those that specifically bindepitopes of 161P2F10B exposed on the cell surface and thus are useful tomodulate growth and survival of cells expressing 161P2F10B by disruptingthe function of a cell expressing 161P2F10B and/or disrupting theinteraction of cells expressing 161P2F10B and the ligand for 161P2F10B.

Preferred antibodies which form one aspect of the invention include butare not limited to antibodies entitled X41(4)6, X41(3)17, X41(3)50,X41(3)15, X41(3)29 and X41(3)37 secreted by a hybridoma deposited withthe American Type Culture Collection (ATCC; 10801 University Blvd.,Manassas, Va. 20110-2209 USA) on 7 Nov. 2002 and assigned as PatentDeposit Designation No. PTA-4792. Patent Deposit Designation No.PTA-4793, Patent Deposit Designation No. PTA-4791, Patent DepositDesignation No. PTA-4791, and Patent Deposit Designation No. PTA-4791(respectively); and derivatives thereof, the production of which isdescribed herein.

Pathological conditions which are characterized by the presence of161P2F10B expression include, but are not restricted to, neoplasms oftissues such as those listed in Table I. One aspect of the inventionprovides a method of detecting the presence of 161P2F10B. A furtheraspect of the invention provides a method of treatment of conditionscharacterized by the presence of 161P2F10B, comprising administering aneffective amount of an anti 161P2F10B antibody. The administration ofanti-161P2F10B antibody is particularly advantageous in the treatment ofconditions characterized by the presence of 161P2F10B.

The antibodies against 161P2F10B for use according to the invention canbe from any species, and can belong to any immunoglobulin class. Thus,for example, the anti 161P2F10B antibody for use according to theinvention can be an immunoglobulin G (IgG), Immunoglobulin M (IgM),immunoglobulin A (IgA), Immunoglobulin E (IgE) or immunoglobulin D(IgD).

The anti 161P2F10B antibody can be from an animal, for example mammaliamor avian origin, and can be for example of murine, rat or human origin.The antibody can be a whole immunoglobulin, or a fragment thereof, forexample a fragment derived by proteolytic cleavage of a whole antibody,such as F(ab′)2 , Fab′ or Fab fragments or fragments obtained byrecombinant DNA techniques, for example Fv fragments.

Particularly useful antibodies for use according to the inventioninclude humanized or fully human anti 161P2F10B antibodies and fragmentsthereof. These antibodies are produced by any suitable procedureincluding, but not restricted to, mammalian cell and bacterial cellfermentation systems.

The anti 161P2F10B Mabs are prepared by immunological techniquesemploying 161P2F10B antigens. Thus, for example, any suitable host canbe injected (immunized) with a suitable reagent which makes 161P2F10Bavailable as an immunogen. Examples of reagents which make 161P2F10Bavailable as an immunogen are purified protein (e.g. the wholeextra-cellular domain (ecd) or fragments there of), peptides designedusing the full length protein as a template (e.g peptides encompassingthe catalytic domain), DNA vectors encoding all or truncated fragmentsof the ecd, recombinant cells expressing 161P2F10B (e.g. Rat-1, Mouse3T3, Mouse 300.19, and mouse NSO), Cell lines with endogenous 161P2F10Bexpression (e.g. human UT-7) or xenografts (i.e. patient derived clearcell and papillary xenografts).

Immune cells, for example splenocytes or lymphocytes, are recovered fromthe immunized host and immortalized, using for example the method ofKohler et al, Eur. J. Immunol 6, 511 (1976), or their immunoglobulingenes can be isolated and transferred to an appropriate DNA vector forexpression in an appropriate cell type. The resulting cells, generatedby either technique, will be selected to obtain a single genetic lineproducing a single unique type of antibody more commonly known as amonoclonal antibody. Antibody fragments can be produced using techniquessuch as enzymatic digestion of whole antibodies e.g. with pepsin(Parham, J. Immunol 131:2895 (1983)) or papain (Lamoyi and Nisonoff, J.Immunol Meth. 56:235 (1983)), or by recombinant DNA techniques.

Suitable hosts for the production of Mab's to 161P2F10B include mice,rats, hamsters and rabbits. For example, mice are immunized with anumber of different reagents which make 161P2F10B available as a sourceof antigenic material (immunogen). The route and timing if theimmunizations will depend on the source and/or embodiment of theimmunogen. Sources of immunogen for 161P2F10B include, but are notrestricted to, peptide, protein, fusion protein, DNA, RNA, cells or cellmembranes as detailed above. These can be used separately as immunogensor in combination to produce a specific immune reaction to 161P2F10B.The use and application of these various immunogens is described fullyin the accompanying examples.

Example 12 HLA Class I and Class II Binding Assays

HLA class I and class II binding assays using purified HLA molecules areperformed in accordance with disclosed protocols (e.g., PCT publicationsWO 94/20127 and WO 94/03205; Sidney et al., Current Protocols inImmunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995);Sette, et al., Mol. Immunol. 31:813 (1994)). Briefly, purified MHCmolecules (5 to 500 nM) are incubated with various unlabeled peptideinhibitors and 1-10 nM 125I-radiolabeled probe peptides as described.Following incubation, MHC-peptide complexes are separated from freepeptide by gel filtration and the fraction of peptide bound isdetermined. Typically, in preliminary experiments, each MHC preparationis titered in the presence of fixed amounts of radiolabeled peptides todetermine the concentration of HLA molecules necessary to bind 10-20% ofthe total radioactivity. All subsequent inhibition and direct bindingassays are performed using these HLA concentrations.

Since under these conditions [label]<[HLA] and IC50≧[HLA], the measuredIC50 values are reasonable approximations of the true KD values. Peptideinhibitors are typically tested at concentrations ranging from 120 μg/mlto 1.2 ng/ml, and are tested in two to four completely independentexperiments. To allow comparison of the data obtained in differentexperiments, a relative binding figure is calculated for each peptide bydividing the IC50 of a positive control for inhibition by the IC50 foreach tested peptide (typically unlabeled versions of the radiolabeledprobe peptide). For database purposes, and inter-experiment comparisons,relative binding values are compiled. These values can subsequently beconverted back into IC50 nM values by dividing the IC50 nM of thepositive controls for inhibition by the relative binding of the peptideof interest. This method of data compilation is accurate and consistentfor comparing peptides that have been tested on different days, or withdifferent lots of purified MHC.

Binding assays as outlined above may be used to analyze HLA supermotifand/or HLA motif-bearing peptides (see Table IV).

Example 13 Identification of HLA Supermotif- and Motif-Bearing CTLCandidate Epitopes

HLA vaccine compositions of the invention can include multiple epitopes.The multiple epitopes can comprise multiple HLA supermotifs or motifs toachieve broad population coverage. This example illustrates theidentification and confirmation of supermotif- and motif-bearingepitopes for the inclusion in such a vaccine composition. Calculation ofpopulation coverage is performed using the strategy described below.

Computer Searches and Algorithms for Identification of Supermotif and/orMotif-Bearing Epitopes

The searches performed to identify the motif-bearing peptide sequencesin the Example entitled “Antigenicity Profiles” and Tables VIII-XXI andXXII-XLIX employ the protein sequence data from the gene product of161P2F10B set forth in FIGS. 2 and 3, the specific search peptides usedto generate the tables are listed in Table VII.

Computer searches for epitopes bearing HLA Class I or Class IIsupermotifs or motifs are performed as follows. All translated 161P2F10Bprotein sequences are analyzed using a text string search softwareprogram to identify potential peptide sequences containing appropriateHLA binding motifs; such programs are readily produced in accordancewith information in the art in view of known motif/supermotifdisclosures. Furthermore, such calculations can be made mentally.

Identified A2-, A3-, and DR-supermotif sequences are scored usingpolynomial algorithms to predict their capacity to bind to specificHLA-Class I or Class II molecules. These polynomial algorithms accountfor the impact of different amino acids at different positions, and areessentially based on the premise that the overall affinity (or ΔG) ofpeptide-HLA molecule interactions can be approximated as a linearpolynomial function of the type:“ΔG”=a _(1i) ×a _(2i) ×a _(3i . . . ×) a _(ni)

where a_(ji) is a coefficient which represents the effect of thepresence of a given amino acid (j) at a given position (i) along thesequence of a peptide of n amino acids. The crucial assumption of thismethod is that the effects at each position are essentially independentof each other (i.e., independent binding of individual side-chains).When residue j occurs at position i in the peptide, it is assumed tocontribute a constant amount j_(i) to the free energy of binding of thepeptide irrespective of the sequence of the rest of the peptide.

The method of derivation of specific algorithm coefficients has beendescribed in Gulukota et al., J. Mol. Biol. 267:1258-126, 1997; (seealso Sidney et al., Human Immunol. 45:79-93, 1996; and Southwood et al.,J. Immunol. 160:3363-3373, 1998). Briefly, for all i positions, anchorand non-anchor alike, the geometric mean of the average relative binding(ARB) of all peptides carrying j is calculated relative to the remainderof the group, and used as the estimate of ji. For Class II peptides, ifmultiple alignments are possible, only the highest scoring alignment isutilized, following an iterative procedure. To calculate an algorithmscore of a given peptide in a test set, the ARB values corresponding tothe sequence of the peptide are multiplied. If this product exceeds achosen threshold, the peptide is predicted to bind. Appropriatethresholds are chosen as a function of the degree of stringency ofprediction desired.

Selection of HLA-A2 Supertype Cross-Reactive Peptides

Protein sequences from 161P2F10B are scanned utilizing motifidentification software, to identify 8-, 9- 10- and 11-mer sequencescontaining the HLA-A2-supermotif main anchor specificity. Typically,these sequences are then scored using the protocol described above andthe peptides corresponding to the positive-scoring sequences aresynthesized and tested for their capacity to bind purified HLA-A*0201molecules in vitro (HLA-A*0201 is considered a prototype A2 supertypemolecule).

These peptides are then tested for the capacity to bind to additionalA2-supertype molecules (A*0202, A*0203, A*0206, and A*6802). Peptidesthat bind to at least three of the five A2-supertype alleles tested aretypically deemed A2-supertype cross-reactive binders. Preferred peptidesbind at an affinity equal to or less than 500 nM to three or more HLA-A2supertype molecules.

Selection of HLA-A3 Supermotif-Bearing Epitopes

The 161P2F10B protein sequence(s) scanned above is also examined for thepresence of peptides with the HLA-A3-supermotif primary anchors.Peptides corresponding to the HLA A3 supermotif-bearing sequences arethen synthesized and tested for binding to HLA-A*0301 and HLA-A*1101molecules, the molecules encoded by the two most prevalent A3-supertypealleles. The peptides that bind at least one of the two alleles withbinding affinities of ≦500 nM, often ≦200 nM, are then tested forbinding cross-reactivity to the other common A3-supertype alleles (e.g.,A*3101, A*3301, and A*6801) to identify those that can bind at leastthree of the five HLA-A3-supertype molecules tested.

Selection of HLA-B7 Supermotif Bearing Epitopes

The 161P2F10B protein(s) scanned above is also analyzed for the presenceof 8-, 9- 10-, or 11-mer peptides with the HLA-B7-supermotif.Corresponding peptides are synthesized and tested for binding toHLA-B*0702, the molecule encoded by the most common B7-supertype allele(i.e., the prototype B7 supertype allele). Peptides binding B*0702 withIC₅₀ of ≦500 nM are identified using standard methods. These peptidesare then tested for binding to other common B7-supertype molecules(e.g., B*3501, B*5101, B*5301, and B*5401). Peptides capable of bindingto three or more of the five B7-supertype alleles tested are therebyidentified.

Selection of A1 and A24 Motif-Bearing Epitopes

To further increase population coverage, HLA-A1 and -A24 epitopes canalso be incorporated into vaccine compositions. An analysis of the161P2F10B protein can also be performed to identify HLA-A1- andA24-motif-containing sequences.

High affinity and/or cross-reactive binding epitopes that bear othermotif and/or supermotifs are identified using analogous methodology.

Example 14 Confirmation of Immunogenicity

Cross-reactive candidate CTL A2-supermotif-bearing peptides that areidentified as described herein are selected to confirm in vitroimmunogenicity. Confirmation is performed using the followingmethodology:

Target Cell Lines for Cellular Screening:

The .221A2.1 cell line, produced by transferring the HLA-A2.1 gene intothe HLA-A, -B, -C null mutant human B-lymphoblastoid cell line 721.221,is used as the peptide-loaded target to measure activity ofHLA-A2.1-restricted CTL. This cell line is grown in RPMI-1640 mediumsupplemented with antibiotics, sodium pyruvate, nonessential amino acidsand 10% (v/v) heat inactivated FCS. Cells that express an antigen ofinterest, or transfectants comprising the gene encoding the antigen ofinterest, can be used as target cells to confirm the ability ofpeptide-specific CTLs to recognize endogenous antigen.

Primary CTL Induction Cultures:

Generation of Dendritic Cells (DC): PBMCs are thawed in RPMI with 30μg/ml DNAse, washed twice and resuspended in complete medium (RPMI-1640plus 5% AB human serum, non-essential amino acids, sodium pyruvate,L-glutamine and penicillin/streptomycin). The monocytes are purified byplating 10×106 PBMC/well in a 6-well plate. After 2 hours at 37° C., thenon-adherent cells are removed by gently shaking the plates andaspirating the supernatants. The wells are washed a total of three timeswith 3 ml RPMI to remove most of the non-adherent and loosely adherentcells. Three ml of complete medium containing 50 ng/ml of GM-CSF and1,000 U/ml of IL-4 are then added to each well. TNFα is added to the DCson day 6 at 75 ng/ml and the cells are used for CTL induction cultureson day 7.

Induction of CTL with DC and Peptide: CD8+ T-cells are isolated bypositive selection with Dynal immunomagnetic beads (Dynabeads® M-450)and the detacha-bead® reagent. Typically about 200-250×106 PBMC areprocessed to obtain 24×106 CD8+ T-cells (enough for a 48-well plateculture). Briefly, the PBMCs are thawed in RPMI with 30 μg/ml DNAse,washed once with PBS containing 1% human AB serum and resuspended inPBS/1% AB serum at a concentration of 20×106 cells/ml. The magneticbeads are washed 3 times with PBS/AB serum, added to the cells (140μlbeads/20×106 cells) and incubated for 1 hour at 4° C. with continuousmixing. The beads and cells are washed 4× with PBS/AB serum to removethe nonadherent cells and resuspended at 100×106 cells/ml (based on theoriginal cell number) in PBS/AB serum containing 100 μl/ml detacha-bead®reagent and 30 μg/ml DNAse. The mixture is incubated for 1 hour at roomtemperature with continuous mixing. The beads are washed again withPBS/AB/DNAse to collect the CD8+ T-cells. The DC are collected andcentrifuged at 1300 rpm for 5-7 minutes, washed once with PBS with 1%BSA, counted and pulsed with 40 μg/ml of peptide at a cell concentrationof 1-2×106/ml in the presence of 3 μg/ml β2-microglobulin for 4 hours at20° C. The DC are then irradiated (4,200 rads), washed 1 time withmedium and counted again.

Setting up induction cultures: 0.25 ml cytokine-generated DC (at 1×105cells/ml) are co-cultured with 0.25 ml of CD8+ T-cells (at 2×106cell/ml) in each well of a 48-well plate in the presence of 10 ng/ml ofIL-7. Recombinant human IL-10 is added the next day at a finalconcentration of 10 ng/ml and rhuman IL-2 is added 48 hours later at 10IU/ml.

Restimulation of the induction cultures with peptide-pulsed adherentcells: Seven and fourteen days after the primary induction, the cellsare restimulated with peptide-pulsed adherent cells. The PBMCs arethawed and washed twice with RPMI and DNAse. The cells are resuspendedat 5×106 cells/ml and irradiated at ˜4200 rads. The PBMCs are plated at2×106 in 0.5 ml complete medium per well and incubated for 2 hours at37° C. The plates are washed twice with RPMI by tapping the plate gentlyto remove the nonadherent cells and the adherent cells pulsed with 10μg/ml of peptide in the presence of 3 μg/ml β2 microglobulin in 0.25 mlRPMI/5% AB per well for 2 hours at 37° C. Peptide solution from eachwell is aspirated and the wells are washed once with RPMI. Most of themedia is aspirated from the induction cultures (CD8+ cells) and broughtto 0.5 ml with fresh media. The cells are then transferred to the wellscontaining the peptide-pulsed adherent cells. Twenty four hours laterrecombinant human IL-10 is added at a final concentration of 10 ng/mland recombinant human IL2 is added the next day and again 2-3 days laterat 50 IU/ml (Tsai et al., Critical Reviews in Immunology 18(1-2):65-75,1998). Seven days later, the cultures are assayed for CTL activity in a51Cr release assay. In some experiments the cultures are assayed forpeptide-specific recognition in the in situ IFNγ ELISA at the time ofthe second restimulation followed by assay of endogenous recognition 7days later. After expansion, activity is measured in both assays for aside-by-side comparison.

Measurement of CTL Lytic Activity by ⁵¹Cr Release.

Seven days after the second restimulation, cytotoxicity is determined ina standard (5 hr) 51Cr release assay by assaying individual wells at asingle E:T. Peptide-pulsed targets are prepared by incubating the cellswith 10 μg/ml peptide overnight at 37° C.

Adherent target cells are removed from culture flasks with trypsin-EDTA.Target cells are labeled with 200 μCi of 51Cr sodium chromate (Dupont,Wilmington, Del.) for 1 hour at 37° C. Labeled target cells areresuspended at 106 per ml and diluted 1:10 with K562 cells at aconcentration of 3.3×106/ml (an NK-sensitive erythroblastoma cell lineused to reduce non-specific lysis). Target cells (100 μl) and effectors(100 μl) are plated in 96 well round-bottom plates and incubated for 5hours at 37° C. At that time, 100 μl of supernatant are collected fromeach well and percent lysis is determined according to the formula:[(cpm of the test sample-cpm of the spontaneous 51Cr releasesample)/(cpm of the maximal 51Cr release sample-cpm of the spontaneous51Cr release sample)]×100.

Maximum and spontaneous release are determined by incubating the labeledtargets with 1% Triton X-100 and media alone, respectively. A positiveculture is defined as one in which the specific lysis(sample-background) is 10% or higher in the case of individual wells andis 15% or more at the two highest E:T ratios when expanded cultures areassayed.

In Situ Measurement of Human IFNγ Production as an Indicator ofPeptide-Specific and Endogenous Recognition

Immulon 2 plates are coated with mouse anti-human IFNγ monoclonalantibody (4 μg/ml 0.1M NaHCO3, pH8.2) overnight at 4° C. The plates arewashed with Ca2+, Mg2+-free PBS/0.05% Tween 20 and blocked with PBS/10%FCS for two hours, after which the CTLs (100 μl/well) and targets (100μl/well) are added to each well, leaving empty wells for the standardsand blanks (which received media only). The target cells, eitherpeptide-pulsed or endogenous targets, are used at a concentration of1×106 cells/ml. The plates are incubated for 48 hours at 37° C. with 5%CO2.

Recombinant human IFN-gamma is added to the standard wells starting at400 pg or 1200 pg/100 microliter/well and the plate incubated for twohours at 37° C. The plates are washed and 100 μl of biotinylated mouseanti-human IFN-gamma monoclonal antibody (2 microgram/ml in PBS/3%FCS/0.05% Tween 20) are added and incubated for 2 hours at roomtemperature. After washing again, 100 microliter HRP-streptavidin(1:4000) are added and the plates incubated for one hour at roomtemperature. The plates are then washed 6x with wash buffer, 100microliter/well developing solution (TMB 1:1) are added, and the platesallowed to develop for 5-15 minutes. The reaction is stopped with 50microliter/well 1M H3PO4 and read at OD450. A culture is consideredpositive if it measured at least 50 pg of IFN-gamma/well abovebackground and is twice the background level of expression.

CTL Expansion.

Those cultures that demonstrate specific lytic activity againstpeptide-pulsed targets and/or tumor targets are expanded over a two weekperiod with anti-CD3. Briefly, 5×104 CD8+ cells are added to a T25 flaskcontaining the following: 1×106 irradiated (4,200 rad) PBMC (autologousor allogeneic) per ml, 2×105 irradiated (8,000 rad) EBV-transformedcells per ml, and OKT3 (anti-CD3) at 30 ng per ml in RPMI-1640containing 10% (v/v) human AB serum, non-essential amino acids, sodiumpyruvate, 25 μM 2-mercaptoethanol, L-glutamine andpenicillin/streptomycin. Recombinant human IL2 is added 24 hours laterat a final concentration of 200 IU/ml and every three days thereafterwith fresh media at 50 IU/ml. The cells are split if the cellconcentration exceeds 1×106/ml and the cultures are assayed between days13 and 15 at E:T ratios of 30, 10, 3 and 1:1 in the 51Cr release assayor at 1×106/ml in the in situ IFNγ assay using the same targets asbefore the expansion.

Cultures are expanded in the absence of anti-CD3+ as follows. Thosecultures that demonstrate specific lytic activity against peptide andendogenous targets are selected and 5×104 CD8+ cells are added to a T25flask containing the following: 1×106 autologous PBMC per ml which havebeen peptide-pulsed with 10 μg/ml peptide for two hours at 37° C. andirradiated (4,200 rad); 2×105 irradiated (8,000 rad) EBV-transformedcells per ml RPMI-1640 containing 10% (v/v) human AB serum,non-essential AA, sodium pyruvate, 25 mM 2-ME, L-glutamine andgentamicin.

Immunogenicity of A2 Supermotif-Bearing Peptides

A2-supermotif cross-reactive binding peptides are tested in the cellularassay for the ability to induce peptide-specific CTL in normalindividuals. In this analysis, a peptide is typically considered to bean epitope if it induces peptide-specific CTLs in at least individuals,and preferably, also recognizes the endogenously expressed peptide.

Immunogenicity can also be confirmed using PBMCs isolated from patientsbearing a tumor that expresses 161P2F10B. Briefly, PBMCs are isolatedfrom patients, re-stimulated with peptide-pulsed monocytes and assayedfor the ability to recognize peptide-pulsed target cells as well astransfected cells endogenously expressing the antigen.

Evaluation of A*03/A11 Immunogenicity

HLA-A3 supermotif-bearing cross-reactive binding peptides are alsoevaluated for immunogenicity using methodology analogous for that usedto evaluate the immunogenicity of the HLA-A2 supermotif peptides.

Evaluation of B7 Immunogenicity

Immunogenicity screening of the B7-supertype cross-reactive bindingpeptides identified as set forth herein are confirmed in a manneranalogous to the confirmation of A2- and A3-supermotif-bearing peptides.

Peptides bearing other supermotifs/motifs, e.g., HLA-A1, HLA-A24 etc.are also confirmed using similar methodology

Example 15 Implementation of the Extended Supermotif to Improve theBinding Capacity of Native Epitopes by Creating Analogs

HLA motifs and supermotifs (comprising primary and/or secondaryresidues) are useful in the identification and preparation of highlycross-reactive native peptides, as demonstrated herein. Moreover, thedefinition of HLA motifs and supermotifs also allows one to engineerhighly cross-reactive epitopes by identifying residues within a nativepeptide sequence which can be analoged to confer upon the peptidecertain characteristics, e.g. greater cross-reactivity within the groupof HLA molecules that comprise a supertype, and/or greater bindingaffinity for some or all of those HLA molecules. Examples of analogingpeptides to exhibit modulated binding affinity are set forth in thisexample.

Analoging at Primary Anchor Residues

Peptide engineering strategies are implemented to further increase thecross-reactivity of the epitopes. For example, the main anchors ofA2-supermotif-bearing peptides are altered, for example, to introduce apreferred L, I, V, or M at position 2, and I or V at the C-terminus.

To analyze the cross-reactivity of the analog peptides, each engineeredanalog is initially tested for binding to the prototype A2 supertypeallele A*0201, then, if A*0201 binding capacity is maintained, forA2-supertype cross-reactivity.

Alternatively, a peptide is confirmed as binding one or all supertypemembers and then analoged to modulate binding affinity to any one (ormore) of the supertype members to add population coverage.

The selection of analogs for immunogenicity in a cellular screeninganalysis is typically further restricted by the capacity of the parentwild type (WT) peptide to bind at least weakly, i.e., bind at an IC50 of5000 nM or less, to three of more A2 supertype alleles. The rationalefor this requirement is that the WT peptides must be presentendogenously in sufficient quantity to be biologically relevant.Analoged peptides have been shown to have increased immunogenicity andcross-reactivity by T cells specific for the parent epitope (see, e.g.,Parkhurst et al., J. Immunol. 157:2539, 1996; and Pogue et al., Proc.Natl. Acad. Sci. USA 92:8166, 1995).

In the cellular screening of these peptide analogs, it is important toconfirm that analog-specific CTLs are also able to recognize thewild-type peptide and, when possible, target cells that endogenouslyexpress the epitope.

Analoging of HLA-A3 and B7-Supermotif-Bearing Peptides

Analogs of HLA-A3 supermotif-bearing epitopes are generated usingstrategies similar to those employed in analoging HLA-A2supermotif-bearing peptides. For example, peptides binding to 3/5 of theA3-supertype molecules are engineered at primary anchor residues topossess a preferred residue (V, S, M, or A) at position 2.

The analog peptides are then tested for the ability to bind A*03 andA*11 (prototype A3 supertype alleles). Those peptides that demonstrate≦500 nM binding capacity are then confirmed as having A3-supertypecross-reactivity.

Similarly to the A2- and A3-motif bearing peptides, peptides binding 3or more B7-supertype alleles can be improved, where possible, to achieveincreased cross-reactive binding or greater binding affinity or bindinghalf life. B7 supermotif-bearing peptides are, for example, engineeredto possess a preferred residue (V, I, L, or F) at the C-terminal primaryanchor position, as demonstrated by Sidney et al. (J. Immunol.157:3480-3490, 1996).

Analoging at primary anchor residues of other motif and/orsupermotif-bearing epitopes is performed in a like manner

The analog peptides are then be confirmed for immunogenicity, typicallyin a cellular screening assay. Again, it is generally important todemonstrate that analog-specific CTLs are also able to recognize thewild-type peptide and, when possible, targets that endogenously expressthe epitope.

Analoging at Secondary Anchor Residues

Moreover, HLA supermotifs are of value in engineering highlycross-reactive peptides and/or peptides that bind HLA molecules withincreased affinity by identifying particular residues at secondaryanchor positions that are associated with such properties. For example,the binding capacity of a B7 supermotif-bearing peptide with an Fresidue at position 1 is analyzed. The peptide is then analoged to, forexample, substitute L for F at position 1. The analoged peptide isevaluated for increased binding affinity, binding half life and/orincreased cross-reactivity. Such a procedure identifies analogedpeptides with enhanced properties.

Engineered analogs with sufficiently improved binding capacity orcross-reactivity can also be tested for immunogenicity inHLA-B7-transgenic mice, following for example, IFA immunization orlipopeptide immunization. Analoged peptides are additionally tested forthe ability to stimulate a recall response using PBMC from patients with161P2F10B-expressing tumors.

Other Analoging Strategies

Another form of peptide analoging, unrelated to anchor positions,involves the substitution of a cysteine with α-amino butyric acid. Dueto its chemical nature, cysteine has the propensity to form disulfidebridges and sufficiently alter the peptide structurally so as to reducebinding capacity. Substitution of α-amino butyric acid for cysteine notonly alleviates this problem, but has been shown to improve binding andcrossbinding capabilities in some instances (see, e.g., the review bySette et al., In: Persistent Viral Infections, Eds R Ahmed and I. Chen,John Wiley & Sons, England, 1999).

Thus, by the use of single amino acid substitutions, the bindingproperties and/or cross-reactivity of peptide ligands for HLA supertypemolecules can be modulated.

Example 16 Identification and Confirmation of 161P2F10B-DerivedSequences with HLA-DR Binding Motifs

Peptide epitopes bearing an HLA class II supermotif or motif areidentified and confirmed as outlined below using methodology similar tothat described for HLA Class I peptides.

Selection of HLA-DR-Supermotif-Bearing Epitopes.

To identify 161P2F10B-derived, HLA class II HTL epitopes, a 161P2F10Bantigen is analyzed for the presence of sequences bearing anHLA-DR-motif or supermotif. Specifically, 15-mer sequences are selectedcomprising a DR-supermotif, comprising a 9-mer core, and three-residueN- and C-terminal flanking regions (15 amino acids total).

Protocols for predicting peptide binding to DR molecules have beendeveloped (Southwood et al., J. Immunol. 160:3363-3373, 1998). Theseprotocols, specific for individual DR molecules, allow the scoring, andranking, of 9-mer core regions. Each protocol not only scores peptidesequences for the presence of DR-supermotif primary anchors (i.e., atposition 1 and position 6) within a 9-mer core, but additionallyevaluates sequences for the presence of secondary anchors. Usingallele-specific selection tables (see, e.g., Southwood et al., ibid.),it has been found that these protocols efficiently select peptidesequences with a high probability of binding a particular DR molecule.Additionally, it has been found that performing these protocols intandem, specifically those for DR1, DR4w4, and DR7, can efficientlyselect DR cross-reactive peptides.

The 161P2F10B-derived peptides identified above are tested for theirbinding capacity for various common HLA-DR molecules. All peptides areinitially tested for binding to the DR molecules in the primary panel:DR1, DR4w4, and DR7. Peptides binding at least two of these three DRmolecules are then tested for binding to DR2w2 β1, DR2w2 β2, DR6w19, andDR9 molecules in secondary assays. Finally, peptides binding at leasttwo of the four secondary panel DR molecules, and thus cumulatively atleast four of seven different DR molecules, are screened for binding toDR4w15, DR5w11, and DR8w2 molecules in tertiary assays. Peptides bindingat least seven of the ten DR molecules comprising the primary,secondary, and tertiary screening assays are considered cross-reactiveDR binders. 161P2F10B-derived peptides found to bind common HLA-DRalleles are of particular interest.

Selection of DR3 Motif Peptides

Because HLA-DR3 is an allele that is prevalent in Caucasian, Black, andHispanic populations, DR3 binding capacity is a relevant criterion inthe selection of HTL epitopes. Thus, peptides shown to be candidates mayalso be assayed for their DR3 binding capacity. However, in view of thebinding specificity of the DR3 motif, peptides binding only to DR3 canalso be considered as candidates for inclusion in a vaccine formulation.

To efficiently identify peptides that bind DR3, target 161P2F10Bantigens are analyzed for sequences carrying one of the two DR3-specificbinding motifs reported by Geluk et al. (J. Immunol. 152:5742-5748,1994). The corresponding peptides are then synthesized and confirmed ashaving the ability to bind DR3 with an affinity of 1 μM or better, i.e.,less than 1 μM. Peptides are found that meet this binding criterion andqualify as HLA class II high affinity binders.

DR3 binding epitopes identified in this manner are included in vaccinecompositions with DR supermotif-bearing peptide epitopes.

Similarly to the case of HLA class I motif-bearing peptides, the classII motif-bearing peptides are analoged to improve affinity orcross-reactivity. For example, aspartic acid at position 4 of the 9-mercore sequence is an optimal residue for DR3 binding, and substitutionfor that residue often improves DR 3 binding.

Example 17 Immunogenicity of 161P2F10B-Derived HTL Epitopes

This example determines immunogenic DR supermotif- and DR3 motif-bearingepitopes among those identified using the methodology set forth herein.

Immunogenicity of HTL epitopes are confirmed in a manner analogous tothe determination of immunogenicity of CTL epitopes, by assessing theability to stimulate HTL responses and/or by using appropriatetransgenic mouse models Immunogenicity is determined by screening for:1.) in vitro primary induction using normal PBMC or 2.) recall responsesfrom patients who have 161P2F10B-expressing tumors.

Example 18 Calculation of Phenotypic Frequencies of HLA-Supertypes inVarious Ethnic Backgrounds to Determine Breadth of Population Coverage

This example illustrates the assessment of the breadth of populationcoverage of a vaccine composition comprised of multiple epitopescomprising multiple supermotifs and/or motifs.

In order to analyze population coverage, gene frequencies of HLA allelesare determined. Gene frequencies for each HLA allele are calculated fromantigen or allele frequencies utilizing the binomial distributionformulae gf=1-(SQRT(1-af)) (see, e.g., Sidney et al., Human Immunol.45:79-93, 1996). To obtain overall phenotypic frequencies, cumulativegene frequencies are calculated, and the cumulative antigen frequenciesderived by the use of the inverse formula [af=1-(1-Cgf)2].

Where frequency data is not available at the level of DNA typing,correspondence to the serologically defined antigen frequencies isassumed. To obtain total potential supertype population coverage nolinkage disequilibrium is assumed, and only alleles confirmed to belongto each of the supertypes are included (minimal estimates). Estimates oftotal potential coverage achieved by inter-loci combinations are made byadding to the A coverage the proportion of the non-A covered populationthat could be expected to be covered by the B alleles considered (e.g.,total=A+B*(1-A)). Confirmed members of the A3-like supertype are A3,A11, A31, A*3301, and A*6801. Although the A3-like supertype may alsoinclude A34, A66, and A*7401, these alleles were not included in overallfrequency calculations. Likewise, confirmed members of the A2-likesupertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206,A*0207, A*6802, and A*6901. Finally, the B7-like supertype-confirmedalleles are: B7, B*3501-03, B51, B*5301, B*5401, B*5501-2, B*5601,B*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, andB*5602).

Population coverage achieved by combining the A2-, A3- and B7-supertypesis approximately 86% in five major ethnic groups. Coverage may beextended by including peptides bearing the A1 and A24 motifs. Onaverage, A1 is present in 12% and A24 in 29% of the population acrossfive different major ethnic groups (Caucasian, North American Black,Chinese, Japanese, and Hispanic). Together, these alleles arerepresented with an average frequency of 39% in these same ethnicpopulations. The total coverage across the major ethnicities when A1 andA24 are combined with the coverage of the A2-, A3- and B7-supertypealleles is >95%, see, e.g., Table IV (G). An analogous approach can beused to estimate population coverage achieved with combinations of classII motif-bearing epitopes.

Immunogenicity studies in humans (e.g., Bertoni et al., J. Clin. Invest.100:503, 1997; Doolan et al., Immunity 7:97, 1997; and Threlkeld et al.,J. Immunol. 159:1648, 1997) have shown that highly cross-reactivebinding peptides are almost always recognized as epitopes. The use ofhighly cross-reactive binding peptides is an important selectioncriterion in identifying candidate epitopes for inclusion in a vaccinethat is immunogenic in a diverse population.

With a sufficient number of epitopes (as disclosed herein and from theart), an average population coverage is predicted to be greater than 95%in each of five major ethnic populations. The game theory Monte Carlosimulation analysis, which is known in the art (see e.g., Osborne, M. J.and Rubinstein, A. “A course in game theory” MIT Press, 1994), can beused to estimate what percentage of the individuals in a populationcomprised of the Caucasian, North American Black, Japanese, Chinese, andHispanic ethnic groups would recognize the vaccine epitopes describedherein. A preferred percentage is 90%. A more preferred percentage is95%.

Example 19 CTL Recognition Of Endogenously Processed Antigens AfterPriming

This example confirms that CTL induced by native or analoged peptideepitopes identified and selected as described herein recognizeendogenously synthesized, i.e., native antigens.

Effector cells isolated from transgenic mice that are immunized withpeptide epitopes, for example HLA-A2 supermotif-bearing epitopes, arere-stimulated in vitro using peptide-coated stimulator cells. Six dayslater, effector cells are assayed for cytotoxicity and the cell linesthat contain peptide-specific cytotoxic activity are furtherre-stimulated. An additional six days later, these cell lines are testedfor cytotoxic activity on 51Cr labeled Jurkat-A2.1/Kb target cells inthe absence or presence of peptide, and also tested on 51Cr labeledtarget cells bearing the endogenously synthesized antigen, i.e. cellsthat are stably transfected with 161P2F10B expression vectors.

The results demonstrate that CTL lines obtained from animals primed withpeptide epitope recognize endogenously synthesized 161P2F10B antigen.The choice of transgenic mouse model to be used for such an analysisdepends upon the epitope(s) that are being evaluated. In addition toHLA-A*0201/Kb transgenic mice, several other transgenic mouse modelsincluding mice with human A11, which may also be used to evaluate A3epitopes, and B7 alleles have been characterized and others (e.g.,transgenic mice for HLA-A1 and A24) are being developed. HLA-DR1 andHLA-DR3 mouse models have also been developed, which may be used toevaluate HTL epitopes.

Example 20 Activity Of CTL-HTL Conjugated Epitopes in Transgenic Mice

This example illustrates the induction of CTLs and HTLs in transgenicmice, by use of a 161P2F10B-derived CTL and HTL peptide vaccinecompositions. The vaccine composition used herein comprise peptides tobe administered to a patient with a 161P2F10B-expressing tumor. Thepeptide composition can comprise multiple CTL and/or HTL epitopes. Theepitopes are identified using methodology as described herein. Thisexample also illustrates that enhanced immunogenicity can be achieved byinclusion of one or more HTL epitopes in a CTL vaccine composition; sucha peptide composition can comprise an HTL epitope conjugated to a CTLepitope. The CTL epitope can be one that binds to multiple HLA familymembers at an affinity of 500 nM or less, or analogs of that epitope.The peptides may be lipidated, if desired.

Immunization procedures: Immunization of transgenic mice is performed asdescribed (Alexander et al., J. Immunol. 159:4753-4761, 1997). Forexample, A2/Kb mice, which are transgenic for the human HLA A2.1 alleleand are used to confirm the immunogenicity of HLA-A*0201 motif- orHLA-A2 supermotif-bearing epitopes, and are primed subcutaneously (baseof the tail) with a 0.1 ml of peptide in Incomplete Freund's Adjuvant,or if the peptide composition is a lipidated CTL/HTL conjugate, inDMSO/saline, or if the peptide composition is a polypeptide, in PBS orIncomplete Freund's Adjuvant. Seven days after priming, splenocytesobtained from these animals are restimulated with syngenic irradiatedLPS-activated lymphoblasts coated with peptide.

Cell lines: Target cells for peptide-specific cytotoxicity assays areJurkat cells transfected with the HLA-A2.1/Kb chimeric gene (e.g.,Vitiello et al., J. Exp. Med. 173:1007, 1991)

In vitro CTL activation: One week after priming, spleen cells (30×106cells/flask) are co-cultured at 37° C. with syngeneic, irradiated (3000rads), peptide coated lymphoblasts (10×106 cells/flask) in 10 ml ofculture medium/T25 flask. After six days, effector cells are harvestedand assayed for cytotoxic activity.

Assay for cytotoxic activity: Target cells (1.0 to 1.5×106) areincubated at 37° C. in the presence of 200 μl of 51Cr. After 60 minutes,cells are washed three times and resuspended in R10 medium. Peptide isadded where required at a concentration of 1 μg/ml. For the assay, 10451Cr-labeled target cells are added to different concentrations ofeffector cells (final volume of 200 μl) in U-bottom 96-well plates.After a six hour incubation period at 37° C., a 0.1 ml aliquot ofsupernatant is removed from each well and radioactivity is determined ina Micromedic automatic gamma counter. The percent specific lysis isdetermined by the formula: percent specific release=100×(experimentalrelease−spontaneous release)/(maximum release−spontaneous release). Tofacilitate comparison between separate CTL assays run under the sameconditions, % 51Cr release data is expressed as lytic units/106 cells.One lytic unit is arbitrarily defined as the number of effector cellsrequired to achieve 30% lysis of 10,000 target cells in a six hour 51Crrelease assay. To obtain specific lytic units/106, the lytic units/106obtained in the absence of peptide is subtracted from the lyticunits/106 obtained in the presence of peptide. For example, if 30% 51Crrelease is obtained at the effector (E): target (T) ratio of 50:1 (i.e.,5×105 effector cells for 10,000 targets) in the absence of peptide and5:1 (i.e., 5×104 effector cells for 10,000 targets) in the presence ofpeptide, the specific lytic units would be:[(1/50,000)−(1/500,000)]×106=18 LU.

The results are analyzed to assess the magnitude of the CTL responses ofanimals injected with the immunogenic CTL/HTL conjugate vaccinepreparation and are compared to the magnitude of the CTL responseachieved using, for example, CTL epitopes as outlined above in theExample entitled “Confirmation of Immunogenicity.” Analyses similar tothis may be performed to confirm the immunogenicity of peptideconjugates containing multiple CTL epitopes and/or multiple HTLepitopes. In accordance with these procedures, it is found that a CTLresponse is induced, and concomitantly that an HTL response is inducedupon administration of such compositions.

Example 21 Selection of CTL and HTL Epitopes for Inclusion in a161P2F10B-Specific Vaccine

This example illustrates a procedure for selecting peptide epitopes forvaccine compositions of the invention. The peptides in the compositioncan be in the form of a nucleic acid sequence, either single or one ormore sequences (i e, minigene) that encodes peptide(s), or can be singleand/or polyepitopic peptides.

The following principles are utilized when selecting a plurality ofepitopes for inclusion in a vaccine composition. Each of the followingprinciples is balanced in order to make the selection.

Epitopes are selected which, upon administration, mimic immune responsesthat are correlated with 161P2F10B clearance. The number of epitopesused depends on observations of patients who spontaneously clear161P2F10B. For example, if it has been observed that patients whospontaneously clear 161P2F10B-expressing cells generate an immuneresponse to at least three (3) epitopes from 161P2F10B antigen, then atleast three epitopes should be included for HLA class I. A similarrationale is used to determine HLA class II epitopes.

Epitopes are often selected that have a binding affinity of an IC50 of500 nM or less for an HLA class I molecule, or for class II, an IC50 of1000 nM or less; or HLA Class I peptides with high binding scores fromthe BIMAS web site.

In order to achieve broad coverage of the vaccine through out a diversepopulation, sufficient supermotif bearing peptides, or a sufficientarray of allele-specific motif bearing peptides, are selected to givebroad population coverage. In one embodiment, epitopes are selected toprovide at least 80% population coverage. A Monte Carlo analysis, astatistical evaluation known in the art, can be employed to assessbreadth, or redundancy, of population coverage.

When creating polyepitopic compositions, or a minigene that encodessame, it is typically desirable to generate the smallest peptidepossible that encompasses the epitopes of interest. The principlesemployed are similar, if not the same, as those employed when selectinga peptide comprising nested epitopes. For example, a protein sequencefor the vaccine composition is selected because it has maximal number ofepitopes contained within the sequence, i.e., it has a highconcentration of epitopes. Epitopes may be nested or overlapping (i.e.,frame shifted relative to one another). For example, with overlappingepitopes, two 9-mer epitopes and one 10-mer epitope can be present in a10 amino acid peptide. Each epitope can be exposed and bound by an HLAmolecule upon administration of such a peptide. A multi-epitopic,peptide can be generated synthetically, recombinantly, or via cleavagefrom the native source. Alternatively, an analog can be made of thisnative sequence, whereby one or more of the epitopes comprisesubstitutions that alter the cross-reactivity and/or binding affinityproperties of the polyepitopic peptide. Such a vaccine composition isadministered for therapeutic or prophylactic purposes. This embodimentprovides for the possibility that an as yet undiscovered aspect ofimmune system processing will apply to the native nested sequence andthereby facilitate the production of therapeutic or prophylactic immuneresponse-inducing vaccine compositions. Additionally such an embodimentprovides for the possibility of motif-bearing epitopes for an HLA makeupthat is presently unknown. Furthermore, this embodiment (absent thecreating of any analogs) directs the immune response to multiple peptidesequences that are actually present in 161P2F10B, thus avoiding the needto evaluate any junctional epitopes. Lastly, the embodiment provides aneconomy of scale when producing nucleic acid vaccine compositions.Related to this embodiment, computer programs can be derived inaccordance with principles in the art, which identify in a targetsequence, the greatest number of epitopes per sequence length.

A vaccine composition comprised of selected peptides, when administered,is safe, efficacious, and elicits an immune response similar inmagnitude to an immune response that controls or clears cells that bearor overexpress 161P2F10B.

Example 22 Construction of “Minigene” Multi-Epitope DNA Plasmids

This example discusses the construction of a minigene expressionplasmid. Minigene plasmids may, of course, contain variousconfigurations of B cell, CTL and/or HTL epitopes or epitope analogs asdescribed herein.

A minigene expression plasmid typically includes multiple CTL and HTLpeptide epitopes. In the present example, HLA-A2, -A3, -B7supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearingpeptide epitopes are used in conjunction with DR supermotif-bearingepitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearingpeptide epitopes derived 161P2F10B, are selected such that multiplesupermotifs/motifs are represented to ensure broad population coverage.Similarly, HLA class II epitopes are selected from 161P2F10B to providebroad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearingepitopes and HLA DR-3 motif-bearing epitopes are selected for inclusionin the minigene construct. The selected CTL and HTL epitopes are thenincorporated into a minigene for expression in an expression vector.

Such a construct may additionally include sequences that direct the HTLepitopes to the endoplasmic reticulum. For example, the Ii protein maybe fused to one or more HTL epitopes as described in the art, whereinthe CLIP sequence of the Ii protein is removed and replaced with an HLAclass II epitope sequence so that HLA class II epitope is directed tothe endoplasmic reticulum, where the epitope binds to an HLA class IImolecules.

This example illustrates the methods to be used for construction of aminigene-bearing expression plasmid. Other expression vectors that maybe used for minigene compositions are available and known to those ofskill in the art.

The minigene DNA plasmid of this example contains a consensus Kozaksequence and a consensus murine kappa Ig-light chain signal sequencefollowed by CTL and/or HTL epitopes selected in accordance withprinciples disclosed herein. The sequence encodes an open reading framefused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1Myc-His vector.

Overlapping oligonucleotides that can, for example, average about 70nucleotides in length with 15 nucleotide overlaps, are synthesized andHPLC-purified. The oligonucleotides encode the selected peptide epitopesas well as appropriate linker nucleotides, Kozak sequence, and signalsequence. The final multiepitope minigene is assembled by extending theoverlapping oligonucleotides in three sets of reactions using PCR. APerkin/Elmer 9600 PCR machine is used and a total of 30 cycles areperformed using the following conditions: 95° C. for 15 sec, annealingtemperature (5° below the lowest calculated Tm of each primer pair) for30 sec, and 72° C. for 1 min.

For example, a minigene is prepared as follows. For a first PCRreaction, 5 μg of each of two oligonucleotides are annealed andextended: In an example using eight oligonucleotides, i.e., four pairsof primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100μl reactions containing Pfu polymerase buffer (1×=10 mM KCL, 10 mM(NH4)2SO4, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO4, 0.1% Triton X-100,100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. Thefull-length dimer products are gel-purified, and two reactionscontaining the product of 1+2 and 3+4, and the product of 5+6 and 7+8are mixed, annealed, and extended for 10 cycles. Half of the tworeactions are then mixed, and 5 cycles of annealing and extensioncarried out before flanking primers are added to amplify the full lengthproduct. The full-length product is gel-purified and cloned intopCR-blunt (Invitrogen) and individual clones are screened by sequencing.

Example 23 The Plasmid Construct and the Degree to Which it InducesImmunogenicity

The degree to which a plasmid construct, for example a plasmidconstructed in accordance with the previous Example, is able to induceimmunogenicity is confirmed in vitro by determining epitope presentationby APC following transduction or transfection of the APC with anepitope-expressing nucleic acid construct. Such a study determines“antigenicity” and allows the use of human APC. The assay determines theability of the epitope to be presented by the APC in a context that isrecognized by a T cell by quantifying the density of epitope-HLA class Icomplexes on the cell surface. Quantitation can be performed by directlymeasuring the amount of peptide eluted from the APC (see, e.g., Sijts etal., J. Immunol. 156:683-692, 1996; Demotz et al., Nature 342:682-684,1989); or the number of peptide-HLA class I complexes can be estimatedby measuring the amount of lysis or lymphokine release induced bydiseased or transfected target cells, and then determining theconcentration of peptide necessary to obtain equivalent levels of lysisor lymphokine release (see, e.g., Kageyama et al., J. Immunol.154:567-576, 1995).

Alternatively, immunogenicity is confirmed through in vivo injectionsinto mice and subsequent in vitro assessment of CTL and HTL activity,which are analyzed using cytotoxicity and proliferation assays,respectively, as detailed e.g., in Alexander et al., Immunity 1:751-761,1994.

For example, to confirm the capacity of a DNA minigene constructcontaining at least one HLA-A2 supermotif peptide to induce CTLs invivo, HLA-A2.1/Kb transgenic mice, for example, are immunizedintramuscularly with 100 μg of naked cDNA. As a means of comparing thelevel of CTLs induced by cDNA immunization, a control group of animalsis also immunized with an actual peptide composition that comprisesmultiple epitopes synthesized as a single polypeptide as they would beencoded by the minigene.

Splenocytes from immunized animals are stimulated twice with each of therespective compositions (peptide epitopes encoded in the minigene or thepolyepitopic peptide), then assayed for peptide-specific cytotoxicactivity in a 51Cr release assay. The results indicate the magnitude ofthe CTL response directed against the A2-restricted epitope, thusindicating the in vivo immunogenicity of the minigene vaccine andpolyepitopic vaccine.

It is, therefore, found that the minigene elicits immune responsesdirected toward the HLA-A2 supermotif peptide epitopes as does thepolyepitopic peptide vaccine. A similar analysis is also performed usingother HLA-A3 and HLA-B7 transgenic mouse models to assess CTL inductionby HLA-A3 and HLA-B7 motif or supermotif epitopes, whereby it is alsofound that the minigene elicits appropriate immune responses directedtoward the provided epitopes.

To confirm the capacity of a class II epitope-encoding minigene toinduce HTLs in vivo, DR transgenic mice, or for those epitopes thatcross react with the appropriate mouse MHC molecule, I-Ab-restrictedmice, for example, are immunized intramuscularly with 100 μg of plasmidDNA. As a means of comparing the level of HTLs induced by DNAimmunization, a group of control animals is also immunized with anactual peptide composition emulsified in complete Freund's adjuvant.CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunizedanimals and stimulated with each of the respective compositions(peptides encoded in the minigene). The HTL response is measured using a3H-thymidine incorporation proliferation assay, (see, e.g., Alexander etal. Immunity 1:751-761, 1994). The results indicate the magnitude of theHTL response, thus demonstrating the in vivo immunogenicity of theminigene.

DNA minigenes, constructed as described in the previous Example, canalso be confirmed as a vaccine in combination with a boosting agentusing a prime boost protocol. The boosting agent can consist ofrecombinant protein (e.g., Barnett et al., Aids Res. and HumanRetroviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia,for example, expressing a minigene or DNA encoding the complete proteinof interest (see, e.g., Hanke et al., Vaccine 16:439-445, 1998; Sedegahet al., Proc. Natl. Acad. Sci USA 95:7648-53, 1998; Hanke and McMichael,Immunol. Letters 66:177-181, 1999; and Robinson et al., Nature Med.5:526-34, 1999).

For example, the efficacy of the DNA minigene used in a prime boostprotocol is initially evaluated in transgenic mice. In this example,A2.1/Kb transgenic mice are immunized IM with 100 μg of a DNA minigeneencoding the immunogenic peptides including at least one HLA-A2supermotif-bearing peptide. After an incubation period (ranging from 3-9weeks), the mice are boosted IP with 107 pfu/mouse of a recombinantvaccinia virus expressing the same sequence encoded by the DNA minigene.Control mice are immunized with 100 μg of DNA or recombinant vacciniawithout the minigene sequence, or with DNA encoding the minigene, butwithout the vaccinia boost. After an additional incubation period of twoweeks, splenocytes from the mice are immediately assayed forpeptide-specific activity in an ELISPOT assay. Additionally, splenocytesare stimulated in vitro with the A2-restricted peptide epitopes encodedin the minigene and recombinant vaccinia, then assayed forpeptide-specific activity in an alpha, beta and/or gamma IFN ELISA.

It is found that the minigene utilized in a prime-boost protocol elicitsgreater immune responses toward the HLA-A2 supermotif peptides than withDNA alone. Such an analysis can also be performed using HLA-A11 orHLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 orHLA-B7 motif or supermotif epitopes. The use of prime boost protocols inhumans is described below in the Example entitled “Induction of CTLResponses Using a Prime Boost Protocol.”

Example 24 Peptide Compositions for Prophylactic Uses

Vaccine compositions of the present invention can be used to prevent161P2F10B expression in persons who are at risk for tumors that bearthis antigen. For example, a polyepitopic peptide epitope composition(or a nucleic acid comprising the same) containing multiple CTL and HTLepitopes such as those selected in the above Examples, which are alsoselected to target greater than 80% of the population, is administeredto individuals at risk for a 161P2F10B-associated tumor.

For example, a peptide-based composition is provided as a singlepolypeptide that encompasses multiple epitopes. The vaccine is typicallyadministered in a physiological solution that comprises an adjuvant,such as Incomplete Freunds Adjuvant. The dose of peptide for the initialimmunization is from about 1 to about 50,000 μg, generally 100-5,000 μg,for a 70 kg patient. The initial administration of vaccine is followedby booster dosages at 4 weeks followed by evaluation of the magnitude ofthe immune response in the patient, by techniques that determine thepresence of epitope-specific CTL populations in a PBMC sample.Additional booster doses are administered as required. The compositionis found to be both safe and efficacious as a prophylaxis against161P2F10B-associated disease.

Alternatively, a composition typically comprising transfecting agents isused for the administration of a nucleic acid-based vaccine inaccordance with methodologies known in the art and disclosed herein.

Example 25 Polyepitopic Vaccine Compositions Derived from Native161P2F10B Sequences

A native 161P2F10B polyprotein sequence is analyzed, preferably usingcomputer algorithms defined for each class I and/or class II supermotifor motif, to identify “relatively short” regions of the polyprotein thatcomprise multiple epitopes. The “relatively short” regions arepreferably less in length than an entire native antigen. This relativelyshort sequence that contains multiple distinct or overlapping, “nested”epitopes can be used to generate a minigene construct. The construct isengineered to express the peptide, which corresponds to the nativeprotein sequence. The “relatively short” peptide is generally less than250 amino acids in length, often less than 100 amino acids in length,preferably less than 75 amino acids in length, and more preferably lessthan 50 amino acids in length. The protein sequence of the vaccinecomposition is selected because it has maximal number of epitopescontained within the sequence, i.e., it has a high concentration ofepitopes. As noted herein, epitope motifs may be nested or overlapping(i.e., frame shifted relative to one another). For example, withoverlapping epitopes, two 9-mer epitopes and one 10-mer epitope can bepresent in a 10 amino acid peptide. Such a vaccine composition isadministered for therapeutic or prophylactic purposes.

The vaccine composition will include, for example, multiple CTL epitopesfrom 161P2F10B antigen and at least one HTL epitope. This polyepitopicnative sequence is administered either as a peptide or as a nucleic acidsequence which encodes the peptide. Alternatively, an analog can be madeof this native sequence, whereby one or more of the epitopes comprisesubstitutions that alter the cross-reactivity and/or binding affinityproperties of the polyepitopic peptide.

The embodiment of this example provides for the possibility that an asyet undiscovered aspect of immune system processing will apply to thenative nested sequence and thereby facilitate the production oftherapeutic or prophylactic immune response-inducing vaccinecompositions. Additionally, such an embodiment provides for thepossibility of motif-bearing epitopes for an HLA makeup(s) that ispresently unknown. Furthermore, this embodiment (excluding an analogedembodiment) directs the immune response to multiple peptide sequencesthat are actually present in native 161P2F10B, thus avoiding the need toevaluate any junctional epitopes. Lastly, the embodiment provides aneconomy of scale when producing peptide or nucleic acid vaccinecompositions.

Related to this embodiment, computer programs are available in the artwhich can be used to identify in a target sequence, the greatest numberof epitopes per sequence length.

Example 26 Polyepitopic Vaccine Compositions from Multiple Antigens

The 161P2F10B peptide epitopes of the present invention are used inconjunction with epitopes from other target tumor-associated antigens,to create a vaccine composition that is useful for the prevention ortreatment of cancer that expresses 161P2F10B and such other antigens.For example, a vaccine composition can be provided as a singlepolypeptide that incorporates multiple epitopes from 161P2F10B as wellas tumor-associated antigens that are often expressed with a targetcancer associated with 161P2F10B expression, or can be administered as acomposition comprising a cocktail of one or more discrete epitopes.Alternatively, the vaccine can be administered as a minigene constructor as dendritic cells which have been loaded with the peptide epitopesin vitro.

Example 27 Use of Peptides to Evaluate an Immune Response

Peptides of the invention may be used to analyze an immune response forthe presence of specific antibodies, CTL or HTL directed to 161P2F10B.Such an analysis can be performed in a manner described by Ogg et al.,Science 279:2103-2106, 1998. In this Example, peptides in accordancewith the invention are used as a reagent for diagnostic or prognosticpurposes, not as an immunogen.

In this example highly sensitive human leukocyte antigen tetramericcomplexes (“tetramers”) are used for a cross-sectional analysis of, forexample, 161P2F10B HLA-A*0201-specific CTL frequencies from HLAA*0201-positive individuals at different stages of disease or followingimmunization comprising a 161P2F10B peptide containing an A*0201 motif.Tetrameric complexes are synthesized as described (Musey et al., N.Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A*0201in this example) and β2-microglobulin are synthesized by means of aprokaryotic expression system. The heavy chain is modified by deletionof the transmembrane-cytosolic tail and COOH-terminal addition of asequence containing a BirA enzymatic biotinylation site. The heavychain, β2-microglobulin, and peptide are refolded by dilution. The 45-kDrefolded product is isolated by fast protein liquid chromatography andthen biotinylated by BirA in the presence of biotin (Sigma, St. Louis,Mo.), adenosine 5′ triphosphate and magnesium.Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, andthe tetrameric product is concentrated to 1 mg/ml. The resulting productis referred to as tetramer-phycoerythrin.

For the analysis of patient blood samples, approximately one millionPBMCs are centrifuged at 300 g for 5 minutes and resuspended in 50 μl ofcold phosphate-buffered saline. Tri-color analysis is performed with thetetramer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38. ThePBMCs are incubated with tetramer and antibodies on ice for 30 to 60 minand then washed twice before formaldehyde fixation. Gates are applied tocontain >99.98% of control samples. Controls for the tetramers includeboth A*0201-negative individuals and A*0201-positive non-diseaseddonors. The percentage of cells stained with the tetramer is thendetermined by flow cytometry. The results indicate the number of cellsin the PBMC sample that contain epitope-restricted CTLs, thereby readilyindicating the extent of immune response to the 161P2F10B epitope, andthus the status of exposure to 161P2F10B, or exposure to a vaccine thatelicits a protective or therapeutic response.

Example 28 Use of Peptide Epitopes to Evaluate Recall Responses

The peptide epitopes of the invention are used as reagents to evaluate Tcell responses, such as acute or recall responses, in patients. Such ananalysis may be performed on patients who have recovered from161P2F10B-associated disease or who have been vaccinated with a161P2F10B vaccine.

For example, the class I restricted CTL response of persons who havebeen vaccinated may be analyzed. The vaccine may be any 161P2F10Bvaccine. PBMC are collected from vaccinated individuals and HLA typed.Appropriate peptide epitopes of the invention that, optimally, bearsupermotifs to provide cross-reactivity with multiple HLA supertypefamily members, are then used for analysis of samples derived fromindividuals who bear that HLA type.

PBMC from vaccinated individuals are separated on Ficoll-Histopaquedensity gradients (Sigma Chemical Co., St. Louis, Mo.), washed threetimes in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCOLaboratories) supplemented with L-glutamine (2 mM), penicillin (50U/ml), streptomycin (50 μg/ml), and Hepes (10 mM) containing 10%heat-inactivated human AB serum (complete RPMI) and plated usingmicroculture formats. A synthetic peptide comprising an epitope of theinvention is added at 10 μg/ml to each well and HBV core 128-140 epitopeis added at 1 μg/ml to each well as a source of T cell help during thefirst week of stimulation.

In the microculture format, 4×105 PBMC are stimulated with peptide in 8replicate cultures in 96-well round bottom plate in 100 μl/well ofcomplete RPMI. On days 3 and 10, 100 μl of complete RPMI and 20 U/mlfinal concentration of rIL-2 are added to each well. On day 7 thecultures are transferred into a 96-well flat-bottom plate andrestimulated with peptide, rIL-2 and 105 irradiated (3,000 rad)autologous feeder cells. The cultures are tested for cytotoxic activityon day 14. A positive CTL response requires two or more of the eightreplicate cultures to display greater than 10% specific 51Cr release,based on comparison with non-diseased control subjects as previouslydescribed (Rehermann, et al., Nature Med. 2:1104,1108, 1996; Rehermannet al., J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al. J.Clin. Invest. 98:1432-1440, 1996).

Target cell lines are autologous and allogeneic EBV-transformed B-LCLthat are either purchased from the American Society forHistocompatibility and Immunogenetics (ASHI, Boston, Mass.) orestablished from the pool of patients as described (Guilhot, et al. J.Virol. 66:2670-2678, 1992).

Cytotoxicity assays are performed in the following manner. Target cellsconsist of either allogeneic HLA-matched or autologous EBV-transformed Blymphoblastoid cell line that are incubated overnight with the syntheticpeptide epitope of the invention at 10 μM, and labeled with 100 μCi of51Cr (Amersham Corp., Arlington Heights, Ill.) for 1 hour after whichthey are washed four times with HBSS.

Cytolytic activity is determined in a standard 4-h, split well 51Crrelease assay using U-bottomed 96 well plates containing 3,000targets/well. Stimulated PBMC are tested at effector/target (E/T) ratiosof 20-50:1 on day 14. Percent cytotoxicity is determined from theformula: 100×[(experimental release-spontaneous release)/maximumrelease-spontaneous release)]. Maximum release is determined by lysis oftargets by detergent (2% Triton X-100; Sigma Chemical Co., St. Louis,Mo.). Spontaneous release is <25% of maximum release for allexperiments.

The results of such an analysis indicate the extent to whichHLA-restricted CTL populations have been stimulated by previous exposureto 161P2F10B or a 161P2F10B vaccine.

Similarly, Class II restricted HTL responses may also be analyzed.Purified PBMC are cultured in a 96-well flat bottom plate at a densityof 1.5×105 cells/well and are stimulated with 10 μg/ml synthetic peptideof the invention, whole 161P2F10B antigen, or PHA. Cells are routinelyplated in replicates of 4-6 wells for each condition. After seven daysof culture, the medium is removed and replaced with fresh mediumcontaining 10 U/ml IL-2. Two days later, 1 μCi 3H-thymidine is added toeach well and incubation is continued for an additional 18 hours.Cellular DNA is then harvested on glass fiber mats and analyzed for3H-thymidine incorporation. Antigen-specific T cell proliferation iscalculated as the ratio of 3H-thymidine incorporation in the presence ofantigen divided by the 3H-thymidine incorporation in the absence ofantigen.

Example 29 Induction of Specific CTL Response in Humans

A human clinical trial for an immunogenic composition comprising CTL andHTL epitopes of the invention is set up as an IND Phase I, doseescalation study and carried out as a randomized, double-blind,placebo-controlled trial. Such a trial is designed, for example, asfollows:

A total of about 27 individuals are enrolled and divided into 3 groups:

Group I: 3 subjects are injected with placebo and 6 subjects areinjected with 5 μg of peptide composition;

Group II: 3 subjects are injected with placebo and 6 subjects areinjected with 50 μg peptide composition;

Group III: 3 subjects are injected with placebo and 6 subjects areinjected with 500 μg of peptide composition.

After 4 weeks following the first injection, all subjects receive abooster inoculation at the same dosage.

The endpoints measured in this study relate to the safety andtolerability of the peptide composition as well as its immunogenicity.Cellular immune responses to the peptide composition are an index of theintrinsic activity of this the peptide composition, and can therefore beviewed as a measure of biological efficacy. The following summarize theclinical and laboratory data that relate to safety and efficacyendpoints.

Safety: The incidence of adverse events is monitored in the placebo anddrug treatment group and assessed in terms of degree and reversibility.

Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy,subjects are bled before and after injection. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

The vaccine is found to be both safe and efficacious.

Example 30 Phase II Trials in Patients Expressing 161P2F10B

Phase II trials are performed to study the effect of administering theCTL-HTL peptide compositions to patients having cancer that expresses161P2F10B. The main objectives of the trial are to determine aneffective dose and regimen for inducing CTLs in cancer patients thatexpress 161P2F10B, to establish the safety of inducing a CTL and HTLresponse in these patients, and to see to what extent activation of CTLsimproves the clinical picture of these patients, as manifested, e.g., bythe reduction and/or shrinking of lesions. Such a study is designed, forexample, as follows:

The studies are performed in multiple centers. The trial design is anopen-label, uncontrolled, dose escalation protocol wherein the peptidecomposition is administered as a single dose followed six weeks later bya single booster shot of the same dose. The dosages are 50, 500 and5,000 micrograms per injection. Drug-associated adverse effects(severity and reversibility) are recorded.

There are three patient groupings. The first group is injected with 50micrograms of the peptide composition and the second and third groupswith 500 and 5,000 micrograms of peptide composition, respectively. Thepatients within each group range in age from 21-65 and represent diverseethnic backgrounds. All of them have a tumor that expresses 161P2F10B.

Clinical manifestations or antigen-specific T-cell responses aremonitored to assess the effects of administering the peptidecompositions. The vaccine composition is found to be both safe andefficacious in the treatment of 161P2F10B-associated disease.

Example 31 Induction of CTL Responses Using a Prime Boost Protocol

A prime boost protocol similar in its underlying principle to that usedto confirm the efficacy of a DNA vaccine in transgenic mice, such asdescribed above in the Example entitled “The Plasmid Construct and theDegree to Which It Induces Immunogenicity,” can also be used for theadministration of the vaccine to humans. Such a vaccine regimen caninclude an initial administration of, for example, naked DNA followed bya boost using recombinant virus encoding the vaccine, or recombinantprotein/polypeptide or a peptide mixture administered in an adjuvant.

For example, the initial immunization may be performed using anexpression vector, such as that constructed in the Example entitled“Construction of “Minigene” Multi-Epitope DNA Plasmids” in the form ofnaked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also beadministered using a gene gun. Following an incubation period of 3-4weeks, a booster dose is then administered. The booster can berecombinant fowlpox virus administered at a dose of 5−10⁷ to 5×10⁹ pfu.An alternative recombinant virus, such as an MVA, canarypox, adenovirus,or adeno-associated virus, can also be used for the booster, or thepolyepitopic protein or a mixture of the peptides can be administered.For evaluation of vaccine efficacy, patient blood samples are obtainedbefore immunization as well as at intervals following administration ofthe initial vaccine and booster doses of the vaccine. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

Analysis of the results indicates that a magnitude of responsesufficient to achieve a therapeutic or protective immunity against161P2F10B is generated.

Example 32 Administration of Vaccine Compositions Using Dendritic Cells(DC)

Vaccines comprising peptide epitopes of the invention can beadministered using APCs, or “professional” APCs such as DC. In thisexample, peptide-pulsed DC are administered to a patient to stimulate aCTL response in vivo. In this method, dendritic cells are isolated,expanded, and pulsed with a vaccine comprising peptide CTL and HTLepitopes of the invention. The dendritic cells are infused back into thepatient to elicit CTL and HTL responses in vivo. The induced CTL and HTLthen destroy or facilitate destruction, respectively, of the targetcells that bear the 161P2F10B protein from which the epitopes in thevaccine are derived.

For example, a cocktail of epitope-comprising peptides is administeredex vivo to PBMC, or isolated DC therefrom. A pharmaceutical tofacilitate harvesting of DC can be used, such as Progenipoietin™(Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC withpeptides, and prior to reinfusion into patients, the DC are washed toremove unbound peptides.

As appreciated clinically, and readily determined by one of skill basedon clinical outcomes, the number of DC reinfused into the patient canvary (see, e.g., Nature Med. 4:328, 1998; Nature Med. 2:52, 1996 andProstate 32:272, 1997). Although 2−50×10⁶ DC per patient are typicallyadministered, larger number of DC, such as 10⁷ or 10⁸ can also beprovided. Such cell populations typically contain between 50-90% DC.

In some embodiments, peptide-loaded PBMC are injected into patientswithout purification of the DC. For example, PBMC generated aftertreatment with an agent such as Progenipoietin™ are injected intopatients without purification of the DC. The total number of PBMC thatare administered often ranges from 10⁸ to 10¹⁰. Generally, the celldoses injected into patients is based on the percentage of DC in theblood of each patient, as determined, for example, by immunofluorescenceanalysis with specific anti-DC antibodies. Thus, for example, ifProgenipoietin™ mobilizes 2% DC in the peripheral blood of a givenpatient, and that patient is to receive 5×10⁶ DC, then the patient willbe injected with a total of 2.5×10⁸ peptide-loaded PBMC. The percent DCmobilized by an agent such as Progenipoietin™ is typically estimated tobe between 2-10%, but can vary as appreciated by one of skill in theart.

Ex Vivo Activation of CTL/HTL Responses

Alternatively, ex vivo CTL or HTL responses to 161P2F10B antigens can beinduced by incubating, in tissue culture, the patient's, or geneticallycompatible, CTL or HTL precursor cells together with a source of APC,such as DC, and immunogenic peptides. After an appropriate incubationtime (typically about 7-28 days), in which the precursor cells areactivated and expanded into effector cells, the cells are infused intothe patient, where they will destroy (CTL) or facilitate destruction(HTL) of their specific target cells, i.e., tumor cells.

Example 33 An Alternative Method of Identifying and ConfirmingMotif-Bearing Peptides

Another method of identifying and confirming motif-bearing peptides isto elute them from cells bearing defined MHC molecules. For example, EBVtransformed B cell lines used for tissue typing have been extensivelycharacterized to determine which HLA molecules they express. In certaincases these cells express only a single type of HLA molecule. Thesecells can be transfected with nucleic acids that express the antigen ofinterest, e.g. 161P2F10B. Peptides produced by endogenous antigenprocessing of peptides produced as a result of transfection will thenbind to HLA molecules within the cell and be transported and displayedon the cell's surface. Peptides are then eluted from the HLA moleculesby exposure to mild acid conditions and their amino acid sequencedetermined, e.g., by mass spectral analysis (e.g., Kubo et al., J.Immunol. 152:3913, 1994). Because the majority of peptides that bind aparticular HLA molecule are motif-bearing, this is an alternativemodality for obtaining the motif-bearing peptides correlated with theparticular HLA molecule expressed on the cell.

Alternatively, cell lines that do not express endogenous HLA moleculescan be transfected with an expression construct encoding a single HLAallele. These cells can then be used as described, i.e., they can thenbe transfected with nucleic acids that encode 161P2F10B to isolatepeptides corresponding to 161P2F10B that have been presented on the cellsurface. Peptides obtained from such an analysis will bear motif(s) thatcorrespond to binding to the single HLA allele that is expressed in thecell.

As appreciated by one in the art, one can perform a similar analysis ona cell bearing more than one HLA allele and subsequently determinepeptides specific for each HLA allele expressed. Moreover, one of skillwould also recognize that means other than transfection, such as loadingwith a protein antigen, can be used to provide a source of antigen tothe cell.

Example 34 Complementary Polynucleotides

Sequences complementary to the 161P2F10B-encoding sequences, or anyparts thereof, are used to detect, decrease, or inhibit expression ofnaturally occurring 161P2F10B. Although use of oligonucleotidescomprising from about 15 to 30 base pairs is described, essentially thesame procedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using, e.g., OLIGO 4.06software (National Biosciences) and the coding sequence of 161P2F10B. Toinhibit transcription, a complementary oligonucleotide is designed fromthe most unique 5′ sequence and used to prevent promoter binding to thecoding sequence. To inhibit translation, a complementary oligonucleotideis designed to prevent ribosomal binding to a 161P2F10B-encodingtranscript.

Example 35 Purification of Naturally-Occurring or Recombinant 161P2F10BUsing 161P2F10B-Specific Antibodies

Naturally occurring or recombinant 161P2F10B is substantially purifiedby immunoaffinity chromatography using antibodies specific for161P2F10B. An immunoaffinity column is constructed by covalentlycoupling anti-161P2F10B antibody to an activated chromatographic resin,such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After thecoupling, the resin is blocked and washed according to themanufacturer's instructions.

Media containing 161P2F10B are passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of 161P2F10B (e.g., high ionic strength buffers in thepresence of detergent). The column is eluted under conditions thatdisrupt antibody/161P2F10B binding (e.g., a buffer of pH 2 to pH 3, or ahigh concentration of a chaotrope, such as urea or thiocyanate ion), andGCR.P is collected.

Example 36 Identification of Molecules Which Interact with 161P2F10B

161P2F10B, or biologically active fragments thereof, are labeled with121 1 Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem.J. 133:529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled 161P2F10B, washed, andany wells with labeled 161P2F10B complex are assayed. Data obtainedusing different concentrations of 161P2F10B are used to calculate valuesfor the number, affinity, and association of 161P2F10B with thecandidate molecules.

Example 37 In Vivo Assay for 161P2F10B Tumor Growth Promotion

The effect of the 161P2F10B protein on tumor cell growth is evaluated invivo by gene overexpression in tumor-bearing mice. For example, SCIDmice are injected subcutaneously on each flank with 1×10⁶ of either PC3,TSUPR1, or DU145 cells containing tkNeo empty vector or 161P2F10B. Atleast two strategies may be used: (1) Constitutive 161P2F10B expressionunder regulation of a promoter such as a constitutive promoter obtainedfrom the genomes of viruses such as polyoma virus, fowlpox virus (UK2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2),bovine papilloma virus, avian sarcoma virus, cytomegalovirus, aretrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or fromheterologous mammalian promoters, e.g., the actin promoter or animmunoglobulin promoter, provided such promoters are compatible with thehost cell systems, and (2) regulated expression under control of aninducible vector system, such as ecdysone, tet, etc., provided suchpromoters are compatible with the host cell systems. Tumor volume isthen monitored at the appearance of palpable tumors and followed overtime and determines that 161P2F10B-expressing cells grow at a fasterrate and/or tumors produced by 161P2F10B-expressing cells demonstratecharacteristics of altered aggressiveness (e.g. enhanced metastasis,vascularization, reduced responsiveness to chemotherapeutic drugs).

Additionally, mice can be implanted with 1×105 of the same cellsorthotopically to determine that 161P2F10B has an effect on local growthin the prostate and/or on the ability of the cells to metastasize, e.g.,to lungs, lymph nodes, and bone marrow.

The assay is also useful to determine the 161P2F10B-inhibitory effect ofcandidate therapeutic compositions, such as for example, small moleculedrugs, 161P2F10B intrabodies, 161P2F10B antisense molecules andribozymes.

Example 38 161P2F10B Monoclonal Antibody-Mediated Inhibition of ProstateTumors In Vivo

The significant expression of 161P2F10B, in cancer tissues, togetherwith its restricted expression in normal tissues along with its cellsurface expression makes 161P2F10B an excellent target for antibodytherapy. Similarly, 161P2F10B is a target for T cell-basedimmunotherapy. Thus, the therapeutic efficacy of anti-161P2F10B mAbs isevaluated, e.g., in human prostate cancer xenograft mouse models usingandrogen-independent LAPC-4 and LAPC-9 xenografts (Craft, N., et al.,Cancer Res, 1999. 59(19): p. 5030-6), kidney cancer xenografts (AGS-K3,AGS-K6), kidney cancer metastases to lymph node (AGS-K6 met) xenografts,and kidney cancer cell lines transfected with 161P2F10B, such as769P-161P2F10B, A498-161P2F10B.

Antibody efficacy on tumor growth and metastasis formation is studied,e.g., in mouse orthotopic prostate cancer xenograft models and mousekidney xenograft models. The antibodies can be unconjugated, asdiscussed in this Example, or can be conjugated to a therapeuticmodality, as appreciated in the art. Anti-161P2F10B mAbs inhibitformation of both the androgen-dependent LAPC-9 and androgen-independentPC3-161P2F10B tumor xenografts. Anti-161P2F10B mAbs also retard thegrowth of established orthotopic tumors and prolonged survival oftumor-bearing mice. These results indicate the utility of anti-161P2F10BmAbs in the treatment of local and advanced stages of prostate cancer.(See, e.g., Saffran, D., et al., PNAS 10:1073-1078). Similarly,anti-161P2F10B mAbs can inhibit formation of AGS-K3 and AGS-K6 tumors inSCID mice, and prevent or retard the growth A498-161P2F10B tumorxenografts. These results indicate the use of anti-161P2F10B mAbs in thetreatment of prostate and/or kidney cancer.

Administration of the anti-161P2F10B mAbs leads to retardation ofestablished orthotopic tumor growth and inhibition of metastasis todistant sites, resulting in a significant prolongation in the survivalof tumor-bearing mice. These studies indicate that 161P2F10B is anattractive target for immunotherapy and demonstrate the therapeutic useof anti-161P2F10B mAbs for the treatment of local and metastaticprostate cancer. This example demonstrates that unconjugated 161P2F10Bmonoclonal antibodies are effective to inhibit the growth of humanprostate tumor xenografts and human kidney xenografts grown in SCIDmice.

Tumor Inhibition Using Multiple Unconjugated 161P2F10B mAbs

Materials and Methods

161P2F10B Monoclonal Antibodies:

Monoclonal antibodies are raised against 161P2F10B as described in theExample entitled “Generation of 161P2F10B Monoclonal Antibodies (mAbs)”or are obtained commercially, e.g., 97A6 (Coulter Immunotech). Theantibodies are characterized by ELISA, Western blot, FACS, andimmunoprecipitation for their capacity to bind 161P2F10B. Epitopemapping data for the anti-161P2F10B mAbs, as determined by ELISA andWestern analysis, recognize epitopes on the 161P2F10B protein. The 97A6antibody binds to amino acids 393-405 of the 161P2F10B protein shown inFIG. 2 Immunohistochemical analysis of cancer tissues and cells isperformed with these antibodies.

The monoclonal antibodies are purified from ascites or hybridoma tissueculture supernatants by Protein-G Sepharose chromatography, dialyzedagainst PBS, filter sterilized, and stored at −20° C. Proteindeterminations are performed by a Bradford assay (Bio-Rad, Hercules,Calif.). A therapeutic monoclonal antibody or a cocktail comprising amixture of individual monoclonal antibodies is prepared and used for thetreatment of mice receiving subcutaneous or orthotopic injections ofLAPC-9 prostate tumor xenografts.

Cancer Xenografts and Cell Lines

The LAPC-9 xenograft, which expresses a wild-type androgen receptor andproduces prostate-specific antigen (PSA), is passaged in 6- to8-week-old male ICR-severe combined immunodeficient (SCID) mice (TaconicFarms) by s.c. trocar implant (Craft, N., et al., supra). The AGS-K3 andAGS-K6 kidney xenografts are also passaged by subcutaneous implants in6- to 8-week old SCID mice. Single-cell suspensions of tumor cells areprepared as described in Craft, et al. The prostate carcinoma cell linePC3 (American Type Culture Collection) is maintained in RPMIsupplemented with L-glutamine and 10% FBS, and the kidney carcinoma lineA498 (American Type Culture Collection) is maintained in DMEMsupplemented with L-glutamine and 10% FBS.

PC3-161P2F10B and A498-161P2F10B cell populations are generated byretroviral gene transfer as described in Hubert, R. S., et al., STEAP: AProstate-specific Cell-surface Antigen Highly Expressed in HumanProstate Tumors, Proc Natl Acad Sci USA, 1999. 96(25): p. 14523-8.Anti-161P2F10B staining is detected by using an FITC-conjugated goatanti-mouse antibody (Southern Biotechnology Associates) followed byanalysis on a Coulter Epics-XL f low cytometer.

Xenograft Mouse Models.

Subcutaneous (s.c.) tumors are generated by injection of 1×10⁶ LAPC-9,AGS-K3, AGS-K6, PC3, PC3-161P2F10B, A498 or A498-161P2F10B cells mixedat a 1:1 dilution with Matrigel (Collaborative Research) in the rightflank of male SCID mice. To test antibody efficacy on tumor formation,i.p. antibody injections are started on the same day as tumor-cellinjections. As a control, mice are injected with either purified mouseIgG (ICN) or PBS; or a purified monoclonal antibody that recognizes anirrelevant antigen not expressed in human cells. In preliminary studies,no difference is found between mouse IgG or PBS on tumor growth. Tumorsizes are determined by vernier caliper measurements, and the tumorvolume is calculated as length x width x height. Mice with s.c. tumorsgreater than 1.5 cm in diameter are sacrificed. PSA levels aredetermined by using a PSA ELISA kit (Anogen, Mississauga, Ontario).Circulating levels of anti-161P2F10B mAbs are determined by a captureELISA kit (Bethyl Laboratories, Montgomery, Tex.). (See, e.g., (Saffran,D., et al., PNAS 10:1073-1078.)

Orthotopic prostate injections are performed under anesthesia by usingketamine/xylazine. For prostate orthotopic studies, an incision is madethrough the abdominal muscles to expose the bladder and seminalvesicles, which then are delivered through the incision to expose thedorsal prostate. LAPC-9 cells (5×10⁵) mixed with Matrigel are injectedinto each dorsal lobe in a 10-μl volume. To monitor tumor growth, miceare bled on a weekly basis for determination of PSA levels. For kidneyorthopotic models, an incision is made through the abdominal muscles toexpose the kidney. AGS-K3 or AGS-K6 cells mixed with Matrigel areinjected under the kidney capsule. The mice are segregated into groupsfor the appropriate treatments, with anti-161P2F10B or control mAbsbeing injected i.p.

Anti-161P2F10B mAbs Inhibit Growth of 161P2F10B-ExpressingXenograft-Cancer Tumors

The effect of anti-161P2F10B mAbs on tumor formation is tested by usingLAPC-9 and/or AGS-K3 orthotopic models. As compared with the s.c. tumormodel, the orthotopic model, which requires injection of tumor cellsdirectly in the mouse prostate or kidney, respectively, results in alocal tumor growth, development of metastasis in distal sites,deterioration of mouse health, and subsequent death (Saffran, D., etal., PNAS supra; Fu, X., et al., Int J Cancer, 1992. 52(6): p. 987-90;Kubota, T., J Cell Biochem, 1994. 56(1): p. 4-8). The features make theorthotopic model more representative of human disease progression andallowed for tracking of the therapeutic effect of mAbs on clinicallyrelevant end points.

Accordingly, tumor cells are injected into the mouse prostate or kidney,and 2 days later, the mice are segregated into two groups and treatedwith either: a) 200-500 μg, of anti-161P2F10B Ab, or b) PBS three timesper week for two to five weeks.

A major advantage of the orthotopic prostate-cancer model is the abilityto study the development of metastases. Formation of metastasis in micebearing established orthotopic tumors is studies by IHC analysis on lungsections using an antibody against a prostate-specific cell-surfaceprotein STEAP expressed at high levels in LAPC-9 xenografts (Hubert, R.S., et al., Proc Natl Acad Sci USA, 1999. 96(25): p. 14523-8) oranti-G250 antibody for kidney cancer models.

Mice bearing established orthotopic LAPC-9 tumors are administered 1000μg injections of either anti-161P2F10B mAb or PBS over a 4-week period.Mice in both groups are allowed to establish a high tumor burden (PSAlevels greater than 300 ng/ml), to ensure a high frequency of metastasisformation in mouse lungs. Mice then are killed and their prostate/kidneyand lungs are analyzed for the presence of tumor cells by IHC analysis.

These studies demonstrate a broad anti-tumor efficacy of anti-161P2F10Bantibodies on initiation and progression of prostate and kidney cancerin xenograft mouse models. Anti-161P2F10B antibodies inhibit tumorformation of both androgen-dependent and androgen-independent prostatetumors as well as retarding the growth of already established tumors andprolong the survival of treated mice. Moreover, anti-161P2F10B mAbsdemonstrate a dramatic inhibitory effect on the spread of local prostatetumor to distal sites, even in the presence of a large tumor burden.Similar therapeutic effects are seen in the kidney cancer model. Thus,anti-161P2F10B mAbs are efficacious on major clinically relevant endpoints (tumor growth), prolongation of survival, and health.

Example 39 Therapeutic and Diagnostic use of Anti-161P2F10B Antibodiesin Humans

Anti-161P2F10B monoclonal antibodies are safely and effectively used fordiagnostic, prophylactic, prognostic and/or therapeutic purposes inhumans Western blot and immunohistochemical analysis of cancer tissuesand cancer xenografts with anti-161P2F10B mAb show strong extensivestaining in carcinoma but significantly lower or undetectable levels innormal tissues. Detection of 161P2F10B in carcinoma and in metastaticdisease demonstrates the usefulness of the mAb as a diagnostic and/orprognostic indicator. Anti-161P2F10B antibodies are therefore used indiagnostic applications such as immunohistochemistry of kidney biopsyspecimens to detect cancer from suspect patients.

As determined by flow cytometry, anti-161P2F10B mAb specifically bindsto carcinoma cells. Thus, anti-161P2F10B antibodies are used indiagnostic whole body imaging applications, such asradioimmunoscintigraphy and radioimmunotherapy, (see, e.g., PotamianosS., et. al. Anticancer Res 20(2A):925-948 (2000)) for the detection oflocalized and metastatic cancers that exhibit expression of 161P2F10B.Shedding or release of an extracellular domain of 161P2F10B into theextracellular milieu, such as that seen for alkaline phosphodiesteraseB10 (Meerson, N. R., Hepatology 27:563-568 (1998)), allows diagnosticdetection of 161P2F10B by anti-161P2F10B antibodies in serum and/orurine samples from suspect patients.

Anti-161P2F10B antibodies that specifically bind 161P2F10B are used intherapeutic applications for the treatment of cancers that express161P2F10B. Anti-161P2F10B antibodies are used as an unconjugatedmodality and as conjugated form in which the antibodies are attached toone of various therapeutic or imaging modalities well known in the art,such as a prodrugs, enzymes or radioisotopes. In preclinical studies,unconjugated and conjugated anti-161P2F10B antibodies are tested forefficacy of tumor prevention and growth inhibition in the SCID mousecancer xenograft models, e.g., kidney cancer models AGS-K3 and AGS-K6,(see, e.g., the Example entitled “161P2F10B Monoclonal Antibody-mediatedInhibition of Bladder and Lung Tumors In Vivo”). Either conjugated andunconjugated anti-161P2F10B antibodies are used as a therapeuticmodality in human clinical trials either alone or in combination withother treatments as described in following Examples.

Example 40 Human Clinical Trials for the Treatment and Diagnosis ofHuman Carcinomas through use of Human Anti-161P2F10B Antibodies In Vivo

Antibodies are used in accordance with the present invention whichrecognize an epitope on 161P2F10B, and are used in the treatment ofcertain tumors such as those listed in Table I. Based upon a number offactors, including 161P2F10B expression levels, tumors such as thoselisted in Table I are presently preferred indications. In connectionwith each of these indications, three clinical approaches aresuccessfully pursued.

I.) Adjunctive therapy: In adjunctive therapy, patients are treated withanti-161P2F10B antibodies in combination with a chemotherapeutic orantineoplastic agent and/or radiation therapy. Primary cancer targets,such as those listed in Table I, are treated under standard protocols bythe addition anti-161P2F10B antibodies to standard first and second linetherapy. Protocol designs address effectiveness as assessed by reductionin tumor mass as well as the ability to reduce usual doses of standardchemotherapy. These dosage reductions allow additional and/or prolongedtherapy by reducing dose-related toxicity of the chemotherapeutic agent.Anti-161P2F10B antibodies are utilized in several adjunctive clinicaltrials in combination with the chemotherapeutic or antineoplastic agentsadriamycin (advanced prostrate carcinoma), cisplatin (advanced head andneck and lung carcinomas), taxol (breast cancer), and doxorubicin(preclinical).

II.) Monotherapy: In connection with the use of the anti-161P2F10Bantibodies in monotherapy of tumors, the antibodies are administered topatients without a chemotherapeutic or antineoplastic agent. In oneembodiment, monotherapy is conducted clinically in end stage cancerpatients with extensive metastatic disease. Patients show some diseasestabilization. Trials demonstrate an effect in refractory patients withcancerous tumors.

III.) Imaging Agent: Through binding a radionuclide (e.g., iodine oryttrium (I131, Y90) to anti-161P2F10B antibodies, the radiolabeledantibodies are utilized as a diagnostic and/or imaging agent. In such arole, the labeled antibodies localize to both solid tumors, as well as,metastatic lesions of cells expressing 161P2F10B. In connection with theuse of the anti-161P2F10B antibodies as imaging agents, the antibodiesare used as an adjunct to surgical treatment of solid tumors, as both apre-surgical screen as well as a post-operative follow-up to determinewhat tumor remains and/or returns. In one embodiment, a (111In)-161P2F10B antibody is used as an imaging agent in a Phase I humanclinical trial in patients having a carcinoma that expresses 161P2F10B(by analogy see, e.g., Divgi et al. J. Natl. Cancer Inst. 83:97-104(1991)). Patients are followed with standard anterior and posteriorgamma camera. The results indicate that primary lesions and metastaticlesions are identified

Dose and Route of Administration

As appreciated by those of ordinary skill in the art, dosingconsiderations can be determined through comparison with the analogousproducts that are in the clinic. Thus, anti-161P2F10B antibodies can beadministered with doses in the range of 5 to 400 mg/m 2, with the lowerdoses used, e.g., in connection with safety studies. The affinity ofanti-161P2F10B antibodies relative to the affinity of a known antibodyfor its target is one parameter used by those of skill in the art fordetermining analogous dose regimens. Further, anti-161P2F10B antibodiesthat are fully human antibodies, as compared to the chimeric antibody,have slower clearance; accordingly, dosing in patients with such fullyhuman anti-161P2F10B antibodies can be lower, perhaps in the range of 50to 300 mg/m2, and still remain efficacious. Dosing in mg/m2, as opposedto the conventional measurement of dose in mg/kg, is a measurement basedon surface area and is a convenient dosing measurement that is designedto include patients of all sizes from infants to adults.

Three distinct delivery approaches are useful for delivery ofanti-161P2F10B antibodies. Conventional intravenous delivery is onestandard delivery technique for many tumors. However, in connection withtumors in the peritoneal cavity, such as tumors of the ovaries, biliaryduct, other ducts, and the like, intraperitoneal administration mayprove favorable for obtaining high dose of antibody at the tumor and toalso minimize antibody clearance. In a similar manner, certain solidtumors possess vasculature that is appropriate for regional perfusion.Regional perfusion allows for a high dose of antibody at the site of atumor and minimizes short term clearance of the antibody.

Clinical Development Plan (CDP)

Overview: The CDP follows and develops treatments of anti-161P2F10Bantibodies in connection with adjunctive therapy, monotherapy, and as animaging agent. Trials initially demonstrate safety and thereafterconfirm efficacy in repeat doses. Trails are open label comparingstandard chemotherapy with standard therapy plus anti-161P2F10Bantibodies. As will be appreciated, one criteria that can be utilized inconnection with enrollment of patients is 161P2F10B expression levels intheir tumors as determined by biopsy.

As with any protein or antibody infusion-based therapeutic, safetyconcerns are related primarily to (i) cytokine release syndrome, i.e.,hypotension, fever, shaking, chills; (ii) the development of animmunogenic response to the material (i.e., development of humanantibodies by the patient to the antibody therapeutic, or HAHAresponse); and, (iii) toxicity to normal cells that express 161P2F10B.Standard tests and follow-up are utilized to monitor each of thesesafety concerns. Anti-161P2F10B antibodies are found to be safe uponhuman administration.

Example 41 Human Clinical Trial Adjunctive Therapy with HumanAnti-161P2F10B Antibody and Chemotherapeutic Agent

A phase I human clinical trial is initiated to assess the safety of sixintravenous doses of a human anti-161P2F10B antibody in connection withthe treatment of a solid tumor, e.g., a cancer of a tissue listed inTable I. In the study, the safety of single doses of anti-161P2F10Bantibodies when utilized as an adjunctive therapy to an antineoplasticor chemotherapeutic agent as defined herein, such as, withoutlimitation: cisplatin, topotecan, doxorubicin, adriamycin, taxol, or thelike, is assessed. The trial design includes delivery of six singledoses of an anti-161P2F10B antibody with dosage of antibody escalatingfrom approximately about 25 mg/m² to about 275 mg/m² over the course ofthe treatment in accordance with the following schedule:

Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 mAb Dose 25 75 125 175 225 275mg/m² mg/m² mg/m² mg/m² mg/m² mg/m² Chemotherapy + + + + + + (standarddose)

Patients are closely followed for one-week following each administrationof antibody and chemotherapy. In particular, patients are assessed forthe safety concerns mentioned above: (i) cytokine release syndrome,i.e., hypotension, fever, shaking, chills; (ii) the development of animmunogenic response to the material (i.e., development of humanantibodies by the patient to the human antibody therapeutic, or HAHAresponse); and, (iii) toxicity to normal cells that express 161P2F10B.Standard tests and follow-up are utilized to monitor each of thesesafety concerns. Patients are also assessed for clinical outcome, andparticularly reduction in tumor mass as evidenced by MRI or otherimaging.

The anti-161P2F10B antibodies are demonstrated to be safe andefficacious, Phase II trials confirm the efficacy and refine optimumdosing.

Example 42 Human Clinical Trial: Monotherapy with Human Anti-161P2F10BAntibody

Anti-161P2F10B antibodies are safe in connection with theabove-discussed adjunctive trial, a Phase II human clinical trialconfirms the efficacy and optimum dosing for monotherapy. Such trial isaccomplished, and entails the same safety and outcome analyses, to theabove-described adjunctive trial with the exception being that patientsdo not receive chemotherapy concurrently with the receipt of doses ofanti-161P2F10B antibodies.

Example 43 Human Clinical Trial: Diagnostic Imaging with Anti-161P2F10BAntibody

Once again, as the adjunctive therapy discussed above is safe within thesafety criteria discussed above, a human clinical trial is conductedconcerning the use of anti-161P2F10B antibodies as a diagnostic imagingagent. The protocol is designed in a substantially similar manner tothose described in the art, such as in Divgi et al. J. Natl. CancerInst. 83:97-104 (1991). The antibodies are found to be both safe andefficacious when used as a diagnostic modality.

Example 44 Homology Comparison of 161P2F10B to Known Sequences

The 161P2F10B gene is identical to a previously cloned and sequencedgene, namely ectonucleotide pyrophosphatase/phosphodiesterase 3 (gi4826896) (Jin-Hua P et al, Genomics 1997, 45:412), also known asphosphodiesterase-I beta; gp130RB13-6; E-NPP3 (ENPP3), PDNP3 and CD203c.The 161P2F10B protein shows 100% identity to human ectonucleotidepyrophosphatase/phosphodiesterase 3 (gi 4826896), and 81% homology and89% identity to rat alkaline phosphodiesterase (gi 1699034). The161P2F10B protein consists of 875 amino acids, with calculated molecularweight of 100.09 kDa, and pI of 6.12. 161P2F10B is a cell surfaceprotein as shown by immunostaining in basophils (Buhring H J et al,Blood 2001, 97:3303) and in epithelial tumor cells as shown in theexample entitled “Expression of 161P2F10B protein in kidney cancerxenograft tissues”. Some localization to the golgi-endoplasmic fractionhas also been observed (Geoffroy V et al, Arch Biochem Biophys. 2001,387:154). Two isoforms of phosphodiesterase 3 have been identified, withone protein containing an additional 145 aa at its amino-terminus (ChoiY H et al, Biochem J. 2001, 353:41). In addition, two variants of161P2F10B have been identified. The first variant contains a pointmutation at amino acid 122 of the 161P2F10B protein, changing a lysineto an arginine at that position. The second variant contains a singlenucleotide polymorphisms, identified at position 383, resulting in achange in amino acid from threonine to proline at that position see URLlocated on the World Wide Web at (FIGS. 4A and 4B). In addition, we haverecently identified another variant of 161P2F10B, namely 161P2F10B v.7.This variant differs from v.1 at its N-terminus as it lacks the first 34aa found in v.1. The loss of the N-terminal 34 aa affects thelocalization of 161P2F10B v.7. PSort analysis reveled that, while161P2F10B v.1 is primarily located at the plasma membrane, 161P2F10B v.7primarily localizes to the cytoplasm (52%) with some localization to thenucleus (17%).

Motif analysis revealed the presence of several known motifs, including2-3 somatostatin B domains located at the amino terminus of the161P2F10B protein, a phosphodiesterase domain and an endonuclease domainat the C-terminus 161P2F10B belongs to a family of closely relatedphosphodiesterases, consisting of PDNP1, -2, and -3 (Bollen M et al,Crit. Rev. Biochem Mol. Biol. 2000, 35: 393). All three members of thisfamily are type II proteins, with a short N-terminus domain locatedintracellularly. They contain one transmembrane domain, a catalyticphosphodiesterase domain and a C-terminal nuclease domain.

Phosphodiesterase 3 expression has been detected in human neoplasticsubmandibular cells, glioma cells, and tansformed lymphocytes (Murata Tet al, Anticancer Drugs 2001, 12:79; Andoh K et al, Biochim Biophys Acta1999, 1446:213; Ekholm D et al, Biochem Pharmacol 1999, 58: 935).

Phosphodiesterase 3 plays an important role in several biologicalprocesses, including release of nucleotides, cell differentiation,metabolism, cell growth, survival, angiogenesis and cell motility(Bollen M et al, Crit. Rev. Biochem Mol. Biol. 2000, 35: 393; Rawadi Get al, Endocrinol 2001, 142:4673; DeFouw L et al, Microvasc Res 2001,62:263). In addition, Phosphodiesterase 3 regulates gene expression inepithelial cells, including the expression of key adhesion moleculessuch as VCAM-1 (Blease K et al, Br J Pharmacol. 1998, 124:229).

This information indicates that 161P2F10B plays a role in the growth ofmammalian cells, supports cell survival and motility, and regulate genetranscription by regulating events in the nucleus. Accordingly, when161P2F10B functions as a regulator of cell transformation, tumorformation, or as a modulator of transcription involved in activatinggenes associated with inflammation, tumorigenesis or proliferation,161P2F10B is used for therapeutic, diagnostic, prognostic and/orpreventative purposes. In addition, when a molecule, such as a avariant, polymorphism or SNP of 161P2F110B is expressed in canceroustissues, such as those listed in Table I, they are used for therapeutic,diagnostic, prognostic and/or preventative purposes.

Example 45 Regulation of Transcription

The cell surface localization of 161P2F10B and ability to regulate VCAMexpression indicate that 161P2F10B is effectively used as a modulator ofthe transcriptional regulation of eukaryotic genes. Regulation of geneexpression is confirmed, e.g., by studying gene expression in cellsexpressing or lacking 161P2F10B. For this purpose, two types ofexperiments are performed.

In the first set of experiments, RNA from parental and161P2F10B-expressing cells are extracted and hybridized to commerciallyavailable gene arrays (Clontech) (Smid-Koopman E et al. Br J Cancer.2000. 83:246). Resting cells as well as cells treated with FBS orandrogen are compared. Differentially expressed genes are identified inaccordance with procedures known in the art. The differentiallyexpressed genes are then mapped to biological pathways (Chen K et al.Thyroid. 2001. 11:41.).

In the second set of experiments, specific transcriptional pathwayactivation is evaluated using commercially available (Stratagene)luciferase reporter constructs including: NFkB-luc, SRE-luc, ELK1-luc,ARE-luc, p53-luc, and CRE-luc. These transcriptional reporters containconsensus binding sites for known transcription factors that liedownstream of well-characterized signal transduction pathways, andrepresent a good tool to ascertain pathway activation and screen forpositive and negative modulators of pathway activation.

Thus, 161P2F10B plays a role in gene regulation, and it is used as atarget for diagnostic, prognostic, preventative and/or therapeuticpurposes.

Example 46 Identification and Confirmation of Potential SignalTransduction Pathways

Many mammalian proteins have been reported to interact with signalingmolecules and to participate in regulating signaling pathways. (JNeurochem. 2001; 76:217-223). In particular, GPCRs have been reported toactivate MAK cascades as well as G proteins, and been associated withthe EGFR pathway in epithelial cells (Naor, Z., et al, Trends EndocrinolMetab. 2000, 11:91; Vacca F et al, Cancer Res. 2000, 60:5310; DellaRocca G J et al, J Biol Chem. 1999, 274:13978). Usingimmunoprecipitation and Western blotting techniques, proteins areidentified that associate with 161P2F10B and mediate signaling events.Several pathways known to play a role in cancer biology can be regulatedby 161P2F10B, including phospholipid pathways such as PI3K, AKT, etc,adhesion and migration pathways, including FAK, Rho, Rac-1, etc, as wellas mitogenic/survival cascades such as ERK, p38, etc (Cell GrowthDiffer. 2000, 11:279; J Biol Chem. 1999, 274:801; Oncogene. 2000,19:3003, J. Cell Biol. 1997, 138:913.).

To confirm that 161P2F10B directly or indirectly activates known signaltransduction pathways in cells, luciferase (luc) based transcriptionalreporter assays are carried out in cells expressing individual genes.These transcriptional reporters contain consensus-binding sites forknown transcription factors that lie downstream of well-characterizedsignal transduction pathways. The reporters and examples of theseassociated transcription factors, signal transduction pathways, andactivation stimuli are listed below.

NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress

SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation

AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress

ARE-luc, androgen receptor; steroids/MAPK;growth/differentiation/apoptosis

p53-luc, p53; SAPK; growth/differentiation/apoptosis

CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress

Gene-mediated effects can be assayed in cells showing mRNA expression.Luciferase reporter plasmids can be introduced by lipid-mediatedtransfection (TFX-50, Promega). Luciferase activity, an indicator ofrelative transcriptional activity, is measured by incubation of cellextracts with luciferin substrate and luminescence of the reaction ismonitored in a luminometer.

Signaling pathways activated by 161P2F10B are mapped and used for theidentification and validation of therapeutic targets. When 161P2F10B isinvolved in cell signaling, it is used as target for diagnostic,prognostic, preventative and/or therapeutic purposes.

Example 47 Involvement in Tumor Progression

The 161P2F10B gene can contribute to the growth of cancer cells. Therole of 161P2F10B in tumor growth is confirmed in a variety of primaryand transfected cell lines including prostate, colon, bladder and kidneycell lines, as well as NIH 3T3 cells engineered to stably express161P2F10B. Parental cells lacking 161P2F10B and cells expressing161P2F10B are evaluated for cell growth using a well-documentedproliferation assay (Fraser S P, Grimes J A, Djamgoz M B. Prostate.2000;44:61, Johnson D E, Ochieng J, Evans S L. Anticancer Drugs. 1996,7:288).

To confirm the role of 161P2F10B in the transformation process, itseffect in colony forming assays is investigated. Parental NIH-3T3 cellslacking 161P2F10B are compared to NIH-3T3 cells expressing 161P2F10B,using a soft agar assay under stringent and more permissive conditions(Song Z. et al. Cancer Res. 2000;60:6730).

To confirm the role of 161P2F10B in invasion and metastasis of cancercells, a well-established assay is used, e.g., a Transwell Insert Systemassay (Becton Dickinson) (Cancer Res. 1999; 59:6010). Control cells,including prostate, colon, bladder and kidney cell lines lacking161P2F10B are compared to cells expressing 161P2F10B. Cells are loadedwith the fluorescent dye, calcein, and plated in the top well of theTranswell insert coated with a basement membrane analog. Invasion isdetermined by fluorescence of cells in the lower chamber relative to thefluorescence of the entire cell population. Using this approach we havedemonstrated that 161P2F10B induces the invasion of 3T3 cells throughthe basement membrane analog matrigel (FIG. 22). As shown in FIG. 22,3T3-neo cells that do not express 161P2F10B exhibit negligible levels ofinvasion though matrigel. Compared to 3T3-neo cells, 3T3-161P2F10Bcells, which express abundant levels of 161P2F10B (FIG. 16), invadethrough matrigel and migrate to the lower chamber of the transwellsystem in a manner similar to that observed with cells expressing thestrong protooncogene 12V-Ras.

161P2F10B can also play a role in cell cycle and apoptosis. Parentalcells and cells expressing 161P2F10B are compared for differences incell cycle regulation using a well-established BrdU assay (Abdel-Malek ZA. J Cell Physiol. 1988, 136:247). In short, cells are grown under bothoptimal (full serum) and limiting (low serum) conditions are labeledwith BrdU and stained with anti-BrdU Ab and propidium iodide. Cells areanalyzed for entry into the G1, S, and G2M phases of the cell cycle.

In contrast to normal cells, cancer cells have been shown to withstandstress, growth factor deprivation and pro-apoptotic signals, therebyproviding tumors with a growth and survival advantage. The effect ofstress on apoptosis is evaluated in control parental cells and cellsexpressing 161P2F10B, including normal and tumor prostate, colon andlung cells using standard assays methods including annexin V binding andcaspase activation (Moore A, et al, Methods Cell Biol. 1998;57:265;Porter A G, Janicke R U. Cell Death Differ. 1999; 6:99). Engineered andparental cells were treated with various chemotherapeutic agents, suchas etoposide, doxorubicin, kinase inhibitors such as staurosporine, DNAdamaging agents such as UV, hypoxia and protein synthesis inhibitors,such as cycloheximide. Cells were stained with annexin V-FITC and celldeath measured by FACS analysis. FIG. 20 shows that expression of161P2F10B prevent the apoptosis of 3T3 cells exposed to staurosporine orUV irradiation. While 64% and 62% of 3T3-neo cells underwent apoptosisin response to staurosporine and UV irradiation, respectively, only 14%and 30% of 161P2F10B-expressing 3T3 cells died under the sameconditions. Similar results were obtained in another experimentcomparing the effect of staurosporine and UV irradiation on 3T3-neocells and clonal populations of 3T3-161P2F10B cell lines (FIG. 19). Aswith the population of 3T3-161P2F10B, clones 3T3-161P2F10B-C and3T3-161P2F10B-10B were resistant to staurosporine-induced cell death.Since caspase activation is a hallmark of apoptosis and serves todistinguish apoptosis from other forms of cell death, we investigatedthe effect of to chemotherapeutic agent and, staurosporine on theapoptosis of kidney cancer cells using caspase activation as assy readout (FIG. 21). The 769 kidney tumor cells that normally lack 161P2F10Bwere engineered to express the 161P2F10B protein as describe in example8, Production of Recombinant 161P2F10B in Higher Eukaryotic Systems,above. The cells were treated with chemotherapeutic agents orstaurosporine, lysed and analyzed for caspase activity. FIG. 21 showsthat expression of 161P2F10B prevents caspase activation in161P2F10B-expressing kidney cancer cells treated with doxorubicin orstaurosporine. These results show that 161P2F10B imparts resistance tothe chemotherapeutic drug doxorubicin and to saurosporine-induced celldeath in kidney cancer cells.

A characteristic that distinguishes cancer cells from normal cells istheir ability to become serum independent and survive in low serumconditions. The effect of serum deprivation on the survival of 161P2F10Bexpressing cells was studied using caspase activation as a read out. Thefibroblast cell line Rat-1 becomes growth arrested when serum deprived,thereby mimicking normal non-transformed cells (James L, Eisenman R N.Proc Natl Acad Sci U S A. 2002, 99:10429). Rat-1 cells expressing c-Myc(Rat-Myc) undergo apoptosis under serum deprivation conditions (James L,Eisenman R N. Proc Natl Acad Sci U S A. 2002, 99:10429). Rat-1 andRat-Myc cells were engineered to stably express 161P2F10B. The cellswere grown in 0.1% or 10% FBS and examined for apoptosis by microscopyand caspase activity (FIGS. 17 and 18). When 161P2F10B is stablyexpressed in Rat-Myc cells, it inhibits Myc-induced apoptosis andreduces caspase activity to background levels. The inhibition of celldeath by 161P2F10B plays a critical role in regulating tumor progressionand tumor load.

When 161P2F10B plays a role in cell growth, transformation, invasion orapoptosis, it is used as a target for diagnostic, prognostic,preventative and/or therapeutic purposes.

Example 48 Involvement in Angiogenesis

Angiogenesis or new capillary blood vessel formation is necessary fortumor growth (Hanahan D, Folkman J. Cell. 1996, 86:353; Folkman J.Endocrinology. 1998 139:441). Based on the effect of phsophodieseteraseinhibitors on endothelial cells, and the homology of 161P2F10B to otherENPP family members, 161P2F10B plays a role in angiogenesis (DeFouw L etal, Microvasc Res 2001, 62:263). Several assays have been developed tomeasure angiogenesis in vitro and in vivo, such as the tissue cultureassays endothelial cell tube formation and endothelial cellproliferation. Using these assays as well as in vitroneo-vascularization, the role of 161P2F10B in angiogenesis, enhancementor inhibition, is confirmed.

For example, endothelial cells engineered to express 161P2F10B areevaluated using tube formation and proliferation assays. The effect of161P2F10B is also confirmed in animal models in vivo. For example, cellseither expressing or lacking 161P2F10B are implanted subcutaneously inimmunocompromised mice. Endothelial cell migration and angiogenesis areevaluated 5-15 days later using immunohistochemistry techniques.Similarly, the secreted extracellular portion of 161P2F10B can functionas an angiogenic factor and enhance the proliferation and tube formationof endothelial cells. The effect of the extracellular domain of161P2F10B on angiogenesis is supported by its similarity to other ENPPs,with biologically active secreted extracellular domain. When 161P2F10Baffects angiogenesis, and it is used as a target for diagnostic,prognostic, preventative and/or therapeutic purposes.

Example 49 Involvement in Protein-Protein Interactions

Several phsophodiesterases have been shown to interact with otherproteins, thereby regulating gene transcription, as well as cell growth(Butt E et al, Mol Pharmacol. 1995, 47:340). Using immunoprecipitationtechniques as well as two yeast hybrid systems, proteins are identifiedthat associate with 161P2F10B Immunoprecipitates from cells expressing161P2F10B and cells lacking 161P2F10B are compared for specificprotein-protein associations.

Studies are performed to confirm the extent of association of 161P2F10Bwith effector molecules, such as nuclear proteins, transcriptionfactors, kinases, phsophates etc. Studies comparing 161P2F10B positiveand 161P2F10B negative cells as well as studies comparingunstimulated/resting cells and cells treated with epithelial cellactivators, such as cytokines, growth factors, androgen andanti-integrin Ab reveal unique interactions.

In addition, protein-protein interactions are confirmed using two yeasthybrid methodology (Curr Opin Chem Biol. 1999, 3:64). A vector carryinga library of proteins fused to the activation domain of a transcriptionfactor is introduced into yeast expressing a 161P2F10B-DNA-bindingdomain fusion protein and a reporter construct. Protein-proteininteraction is detected by colorimetric reporter activity. Specificassociation with effector molecules and transcription factors directsone of skill to the mode of action of 161P2F10B, and thus identifiestherapeutic, prognostic, preventative and/or diagnostic targets forcancer. This and similar assays are also used to identify and screen forsmall molecules that interact with 161P2F10B.

Thus it is found that 161P2F10B associates with proteins and smallmolecules. Accordingly, 161P2F10Band these proteins and small moleculesare used for diagnostic, prognostic, preventative and/or therapeuticpurposes.

Example 50 Involvement in Cell Adhesion

Cell adhesion plays a critical role in tissue colonization andmetastasis. 161P2F10B can participate in cellular organization, and as aconsequence cell adhesion and motility. To confirm that 161P2F10Bregulates cell adhesion, control cells lacking 161P2F10B are compared tocells expressing 161P2F10B, using techniques previously described (see,e.g., Haier et al, Br. J. Cancer. 1999, 80:1867; Lehr and Pienta, J.Natl. Cancer Inst. 1998, 90:118). Briefly, in one embodiment, cellslabeled with a fluorescent indicator, such as calcein, are incubated ontissue culture wells coated with media alone or with matrix proteins.Adherent cells are detected by fluorimetric analysis and percentadhesion is calculated. In another embodiment, cells lacking orexpressing 161P2F10B are analyzed for their ability to mediate cell-celladhesion using similar experimental techniques as described above. Bothof these experimental systems are used to identify proteins, antibodiesand/or small molecules that modulate cell adhesion to extracellularmatrix and cell-cell interaction. Cell adhesion plays a critical role intumor growth, progression, and, colonization, and 161P2F10B is involvedin these processes. Thus, it serves as a diagnostic, prognostic,preventative and/or therapeutic modality.

Example 51 Phosphodiesterase Activity of 161P2F10B ExpressingRecombinant Cell Lines

In order to delineate the function 161P2F10B, several cell lines thatlack 161P2F10B were transduced with 161P2F10B-encoding retovirus asdescribed in example 8, Production of Recombinant 161P2F10B in HigherEukaryotic Systems, above. Cell lines were characterized for 161P2F10Bcell surface expression by FACS analysis (FIGS. 28, 29, 30, and 16).cDNA was stably introduced into the fibroblast lines NIH 3T3 and Rat-1,myeloma NSO cells, and kidney cancer CaKi cells. The cells wereimmunostained with anti-CD203c mAb and analyzed by flow cytometry. FIGS.28, 29, 30, and 16 show that while parental cells fail to express161P2F10B, engineered lines demonstrate abundant expression of 161P2F10Bon their cell surface. Expression of 161P2F10B in engineered cells wascompared to that in UT7, a cell line that expresses 161P2F10Bendogenously (FIG. 28). Our results show that engineered Rat-1 and 3T3cells express 161P2F10B at levels comparable to UT7 cells.

Since 161P2F10B is identical to the ecto-enzyme ENPP3 phosphodieterase,and members of the ENPP family possess pyrophosphatase activities, therecombinant cell lines were also characterized for phosphodiesteraseactivity (FIGS. 28, 29, 30, and 16). Control and 161P2F10B-expressingcells were lysates or intact cells were incubated for at 37 degrees in20 mM Tris/HCL, pH 9.6 containing 5 mM MgCl2 and 1 mM p-nitrophenylthymidine-5′-L-monophosphate. The reaction was terminated by theaddition of 0.1 N NaOH and the reaction product quantified by readingabsorbance at 410 nm FIGS. 28, 29, 30, and 16 show that 161P2F10Bexpression parallels phosphodiesterase activity. Using CaKi cellsexpressing either wild type or mutant 161P2F10B, we show that mutationof T205 inhibits phosphodiesterase activity (FIG. 30). When 161P2F10Bshows phosphodiesterase activity, it is used as a target for diagnostic,prognostic, preventative and/or therapeutic purposes.

In addition to phosphodiesterase activity, members of the ENPP familyexhibit lysophospholipase D (lysoPLD) activity (Umezu-Goto M et al, JCell Biol. 2002, 158: 227). ENPP-2 (aka autotoxin) in particular wasfound to act on lysophosphatidylcholine (LPC) to generatelysophosphatidic acid (LPA) (Umezu-Goto M et al, J Cell Biol. 2002, 158:227; Tokumura A et al, J boil. Chem 2002, 277:39436). LPA is involved invarious biological functions associated with tumor development,including cell proliferation and invasion (Gschwind A, Prenzel N,Ullrich A. Cancer Res. 2002, 62:6329). Based on the homology of161P1F10B to other ENPP family members, 161P2F10B has lysoPLD activity.The lysoPLD activity of 161P2F10B expressing cells is compared to cellslacking 161P2F10B using a standard choline release assay. In short, celllysates are incubated with LPC for 1 hr at 37° C. Liberated choline isdetected by fluoremetry following the addition of choline oxidase. When161P2F10B shows lysoPLD activity, it is used as a target for diagnostic,prognostic, preventative and/or therapeutic purposes.

Example 52 RNA Interference (RNAi)

Several methods of reducing or abolishing the expression of specificgenes have been used for confirming the importance of said genes intumor growth and progression. These methods include antisenseoligonucleotides, morpholino, ribozyme, etc that function in a sequencespecific manner to prevent gene transcription or translation. Morerecently, RNA interference by duplexes of short nucleotide RNAs has beenshown to inhibit gene expression in a sequence specific manner inmammalian cells (Elbashir S et al, Nature 2001, 411:494). RNAinterference (RNAi) makes use of sequence specific double stranded RNAknown as small interfering RNAs (siRNAs) to prevent gene expression.Small interfering RNA (siRNA) are transfected into mammalian cells andthereby mediate sequence specific mRNA degradation. (Elbashir, et al,Nature, 2001; 411: 494). Similarly, siRNA have been used to generatestable vector systems that can be delivered in vitro and in vivo tomammalian cells, thereby providing therapeutic use for siRNAs (Lee N etal, Nature Biotechnol 2002, 19:500).

Several siRNAs can be used to modulate the expression of 161P2F10B inmammalian cells, including for example the following siRNAoligonucleotide sequences:

161P2F10B (1) target: GAAUCUACGUUGACUUUAG (corresponding to nucleotides4-23 of 161P2F10B ORF) (SEQ ID NO: 39)

The sense strand of 161P2F10B (1) can labeled at 3′ with fluorescein,6-FAM (ABS 494 nm, EMM 525 nm, green) for easy detection. The siRNA isdissolved in RNA-free sterile buffer (100 mM KOAc, 30 mM HEPES KOH, 2 mMMOAc, at pH 7.4) to make 20 μM stock (200×). The siRNa is transfectedinto various normal and tumor cells, including UT7, 3T3-161P2F10B,CaKi-161P2F10B and Rat-161P2F10B cells. Control, non-specificoligonucleotide is used as a control to rule out any non-specific effectof 161P2F10B siRNA oligonucleotides

Protein expression is determined 24-48 hours after transfection byimmunostaining followed by flow cytometry. In addition, confirmation ofaltered gene expression is performed by Western blotting. Cellstransfected with control or 161P2F10B-specific siRNAi are compared usingfunctional assays described above, including invasion, proliferation,colony formation and response to apoptotic stimuli. Therefore, the RNAoligonucleotide sequences are used to assess how modulating theexpression of a 161P2F10B gene affects function of cancer cells and/ortissues. Accordingly, the RNA oligonucleotide sequences are used intherapeutic and prophylactic applications.

Example 53 Generation of Antibodies to 161P2F10B Using Peptide Encodingthe Caytalytic Domain of 161P2F10B as the Immunogen

In one embodiment peptides of 22 amino acids encompassing the161P2F10Bcatalytic domain (Threonine (T) at position 205), CGIHSKYMRAMYPTKTFPNHYT(SEQ ID NO: 40) were generated. These were, synthesized and the peptideswere coupled to KLH through the N-terminal cysteine residue.

Balb/c mice were immunized intraperitoneally (i.p.) with 10 μg ofpeptide every 2 weeks over a 4 week period. The initial immunization wasgiven i.p. in Complete Freunds Adjuvant (CFA) and the subsequent twoimmunizations were given i.p. in Incomplete Freunds Adjuvant (IFA).

To determine the specificity of the response following immunization,mice were bled 10 days after the final immunization. Reactivity wasdetermined by Enzyme Linked Immunosorbent Assay (ELISA) using non-KLHconjugated (free) peptide as a target.Mice with the highest titers weregiven a final boost of 10 ng peptide in PBS and sacrificed for fusion 3days later. Spleen cells from the immunized mice were fused with mouseSp2/0 myeloma cells using PEG 1500 according to standard protocols(Kohler et al, Eur J Immunol 6: 511 (1976)). Fused cells were plated in10 96 well microtiter plates and hybridomas were selected using HATmedia supplement. Supernatants from fusion wells were screened 10-17days later by ELISA against 161P2F10B peptide, and clones were thenchecked for the ability of the monoclonal antibody to recognize cellmembrane 161P2F10B by FACS on 161P2F10 expressing Rat-1 cells.

Example 54 Generation of Antibodies to 161P2F10B Using Protein Encodingthe Whole Extra Cellular Domain (aa 1-975) of 161P2F10B as the Immunogen

In one embodiment the whole extra cellular domain of 161P2F10B fused atthe C′ terminal with 6 Histidines (6-His for purification was purifiedhor use as an immunogen.

Balb/c mice were immunized intraperitoneally (i.p.) with 10 μg ofprotein every 2 weeks over a 4 week period. The initial immunization wasgiven i.p. in Complete Freunds Adjuvant (CFA) and the subsequent twoimmunizations were given i.p. in Incomplete Freunds Adjuvant (IFA).

To determine the specificity of the response following immunization,mice were bled 10 days after the final immunization. Reactivity wasdetermined by Enzyme Linked Immunosorbent Assay (ELISA) using purifiedprotein as a screening agent.

Mice with the highest titers were given a final boost of 10 μg proteinin PBS and sacrificed for fusion 3 days later. Spleen cells from theimmunized mice were fused with mouse Sp2/0 myeloma cells using PEG 1500according to standard protocols (Kohler et al, Eur. J. Immunol 6: 511(1976)). Fused cells were plated in 10 96 well microtiter plates andhybridomas were selected using HAT media supplement. Supernatants fromfusion wells were screened 10-17 days later by ELISA against 161P2F10Bprotein, and clones were then checked for the ability of the monoclonalantibody to recognize cell membrane 161P2F10B by FACS on 161P2F10expressing Rat-1 cells.

Example 55 Generation Mabs to 161P2F10B Using DNA Immunization with aVector Encoding 161P2F10B Fused at the C′ Terminus with Human IgG Fc

In another embodiment, a vector was constructed that encodes the 975amino acids of the 161P2F10 extra cellular domain fused at theC-terminus to the human immunoglobulin G1 (IgG) Fc (hinge, CH2, CH3regions). This construct was used in a DNA based immunization strategy.

Balb/c mice were immunized intradermally (ID) at the base of their tail.Three immunizations were given to each mouse of 100 μg of DNA in PBSover a two-week period. To increase the immune response, each mouse wasgiven an i.p. boost of 2 ng of 161P2F10B-Fc protein in tissue culturemedia 10 days after the final DNA immunization. Bleeds were collected 10days after the final immunization and reactivity in the sera to themiddle loop of 161P2F10B was tested by ELISA using 161P2F10B-Fc fusionprotein as a target (test 1). In parallel the sera were also tested onan unrelated human Fc fusion protein (test 2). Specific reactivity tothe 161P2F10B portion of the fusion protein was indicated.

All mice were sacrificed and fusions and hybridoma selection was carriedout as described in Example 54. Hybridoma supernatants were screened10-17 days later by ELISA using 161P2F10B-Fc protein as target.161P2F10B-Fc positives were subsequently cross-screened on irrelevant Fcproteins to identify 161P2F10 specific clones. Monoclonal antibodieswere tested for specificity and reactivity to cell surface 161p2F10Busing recombinant Rat 1 cells. Several antibodies were identified thisway including X41(4)6, X41(3)15, X41(3)17, X41(3)29, X41(3)37 andX41(3)50. These antibodies or binding region thereof secreted by ahybridoma entitled X41(3)15/29/37, X41(4)6, X41(3)17, and X41(3)50 weredeposited with the American Type Culture Collection (ATCC; 10801University Blvd., Manassas, Va. 20110-2209 USA) on 7 Nov. 2002 andassigned as Patent Deposite Designation NO. PTA-4791, Patent DepositeDesignation NO. PTA-4794, Patent Deposite Designation NO. PTA-4792, andPatent Deposite Designation NO. PTA-4793 (respectively). FACS data forthese monoclonal antibodies is shown on (FIG. 40).

Example 56 Generation of Mabs to 161P2F10B Using DNA Immunization with aVector Encoding 161P2F10B Fused at the C′ Terminus with the myc His Tag

In another embodiment, a vector was constructed that encodes the 975amino acids of the 161P2F10 extra cellular domain fused at theC-terminus to the myc-His tag. This construct was used in a DNA basedimmunization strategy.

Balb/c mice were immunized intra-dermally (ID) at the base of theirtail. Three immunizations were given to each mouse of 100 μg of DNA inPBS over a two-week period. To increase the immune response, each mousewas given an i.p. boost of 2 ng of 161P2F10B-Fc protein in tissueculture media 10 days after the final DNA immunization. Bleeds werecollected 10 days after the final immunization and reactivity in thesera to the middle loop of 161P2F10B was tested by ELISA using161P2F10B-Fc fusion protein as a target (test 1). In parallel the serawere also tested on an unrelated human Fc fusion protein (test 2).Specific reactivity to the 161P2F10B portion of the fusion protein wasindicated.

All mice were sacrificed and fusions and hybridoma selection was carriedout as described in Example 11. Hybridoma supernatants were screened10-17 days later by ELISA using 161P2F10B-Fc protein as target.161P2F10B-Fc positives were subsequently cross-screened on irrelevant Fcproteins to identify 161P2F10 specific clones. Monoclonal antibodieswere tested for specificity and reactivity to cell surface 161p2F10Busing recombinant Rat 1 cells.

Example 57 Generation of Monoclonal Antibodies Specific for 161P2F10BUsing UT7 Cells Endogenously Expressing 161P2F10B

It has been reported in the literature that antibodies to 161P2F10B canbe made by immunization with the human erythro-megakaryoblastic cellline UT-7 cultured with IL3 (Buhring et. al. Blood 94(7): 2343. 1999).Antibodies described in this publication are available commercially andhave been used as controls in the invention described here.

In another embodiment, mice were immunized intra-peritoneally with UT-7cells, 106 cells per immunization. A total of 5 immunizations were givenapproximately 2 weeks apart with the final injection being given threedays befor mice were sacrificed for fusions. Mice were bled 10 daysafter the third injection and the 161P2F10B specific titer of the serawas determined by ELISA using 161P2F10 as a screening agent. Mice withhigh titers were then used for fusions as described in Example 11.Monoclonal antibodies generated in this way were selected by ELISA andtheir ability to recognize cells surface 161P2F10B was confirmed by FACSon Rat 1 cells expressing 161P2F10B.

Example 58 Generation of Monoclonal Antibodies Specific for 161P2F10BUsing the Recombinant Cell Line 3T3 Expressing 161P2F10B

In another embodiment, mice were immunized intra-peritoneally with 3T3cells expressing 161P2F10B, 106 cells per immunization. A total of 5immunizations were given approximately 2 weeks apart with the finalinjection being given three days before mice were sacrificed forfusions. Mice were bled 10 days after the third injection and the161P2F10B specific titer of the sera was determined by ELISA using161P2F10 as a screening agent. Mice with high titers were then used forfusions as described in Example 11. Monoclonal antibodies generated inthis way were selected by ELISA and their ability to recognize cellssurface 161P2F10B was confirmed by FACS on Rat 1 cells expressing161P2F10B.

Example 59 Generation of Monoclonal Antibodies Specific for 161P2F10BUsing the Recombinant Cell Line Rat 1 Expressing 161P2F10B

In another embodiment mice were immunized with Rat-1 cells expressing161P2F10B, Mice were then used for fusions as described in Example 11.Monoclonal antibodies generated in this way were selected by ELISA andtheir ability to recognize cells surface 161P2F10B was confirmed by FACSon Rat 1 cells expressing 161P2F10B.

Example 60 Detection of 161P2F10B Protein in Kidney Cancer PatientSpecimens

To confirm the expression of 161P2F10B protein, kidney cancer specimenswere obtained from kidney cancer patients, and stained using thecommercially available antibody 97A6 specific for ENPP3 protein (alsocalled anti-CD203c) (Immunotech, Marseilles, France). Briefly, frozentissues were cut into 4 micron sections and fixed in acetone for 10minutes. The sections were then incubated with PE-labeled mousemonoclonal anti-ENPP3 antibody for 3 hours (FIG. 24A-C), or isotypecontrol antibody (FIG. 44G-I). The slides were washed three times inbuffer, and either analyzed by fluorescence microscopy (FIG. 44A, B andC), or further incubated with DAKO Envision+™ peroxidase-conjugated goatanti-mouse secondary antibody (DAKO Corporation, Carpenteria, Calif.)for 1 hour (FIGS. 44D, E, and F FIG. 24A-C). The sections were thenwashed in buffer, developed using the DAB kit (SIGMA Chemicals),counterstained using hematoxylin, and analyzed by bright fieldmicroscopy (FIGS. 44D, E and F). The results showed strong expression of161P2F10B in the renal carcinoma patient tissue (FIGS. 44A and D) andthe kidney cancer metastasis to lymph node tissue (FIGS. 44C and F), butweakly in normal kidney (FIGS. 44B and E). The expression was detectedmostly around the cell periphery in renal clear cell carcinoma (FIGS.44A and D, FIGS. 24A and B) and was strongly expressed throughout thecells with an apparent predisposition towards the cell periphery inrenal papillary carcinoma (FIG. 24C) indicating that 161P2F10B ismembrane associated in kidney cancer tissues. The weak expressiondetected in normal kidney was localized to the kidney tubules. Thesections stained with the isotype control antibody were negative showingthe specificity of the anti-ENPP3 antibody (FIG. 44G-I). Kidney cancerspecimens were obtained from patients with different types of renaltumor including renal clear cell carcinoma; papillary cell carcinoma;renal cell carcinoma, chromophobe type; transitional cell carcinoma andoncocytoma and were stained for 161P2F10B using the commerciallyavailable antibody 97A6 specific for ENPP3 protein (also calledanti-CD203c) (Immunotech, Marseilles, France). All tissue specimens forrenal clear cell carcinoma and papillary cell carcinoma were positivefor 161P2F10B (Table LIX).

FIG. 45 shows expression of 161P2F10B in human patient cancers byWestern blot analysis. Cell lysates from kidney cancer tissues (KiCa),kidney cancer metastasis to lymph node (KiCa Met), as well as normalkidney (NK) were subjected to western analysis using an anti-161P2F10Bmouse monoclonal antibody. Briefly, tissues (˜25 μg total protein) weresolubilized in SDS-PAGE sample buffer and separated on a 10-20% SDS-PAGEgel and transferred to nitrocellulose. Blots were blocked inTris-buffered saline (TBS)+3% non-fat milk and then probed with purifiedanti-161P2F10B antibody in TBS+0.15% Tween-20+1% milk Blots were thenwashed and incubated with a 1:4,000 dilution of anti-mouse IgG-HRPconjugated secondary antibody. Following washing, anti-161P2F10Bimmunoreactive bands were developed and visualized by enhancedchemiluminescence and exposure to autoradiographic film. The specificanti-161P2F10B immunoreactive bands represent a monomeric form of the161P2F10B protein, which runs at approximately 130 kDa. These resultsdemonstrate that 161P2F10B is useful as a diagnostic and therapeutictarget for kidney cancers, metastatic cancers and other human cancersthat express this protein.

The strong expression of 161P2F10B in kidney cancer tissues and itsrestricted expression in normal kidney as well as its membranelocalization show that 161P2F10B is a target, e.g., for kidney cancerdiagnosis and therapy. The expression detected in kidney cancermetastatic tissue indicates that 161P2F10B is also a target formetastatic disease. As disclosed herein, Western blot andimmunohistochemical analysis of kidney cancer tissues and kidney cancerxenografts with mAb 97A6 showed strong extensive staining of ENPP3 inclear cell kidney carcinoma but significantly lower or undetectablelevels in normal kidney (FIGS. 44, 45, 46, and 24). Detection of161P2F10B (ENPP3) in high grade clear cell carcinoma and in metastaticdisease.

Example 61 Detection of 161P2F10B Protein in Colon Cancer PatientSpecimens

Tissue specimens of colon adenocarcinoma were obtained from ninedifferent colon cancer patients. Frozen tissues were cut into 4 micronsections and fixed in acetone for 10 minutes. The sections were thenincubated with mouse monoclonal anti-ENPP3 antibody (Coulter-Immunotech,Marseilles, France) for 3 hours. The slides were washed three times inbuffer, and further incubated with DAKO Envision+™ peroxidase-conjugatedgoat anti-mouse secondary antibody (DAKO Corporation, Carpenteria,Calif.) for 1 hour. The sections were then washed in buffer, developedusing the DAB kit (SIGMA Chemicals), counterstained using hematoxylin,and analyzed by bright field microscopy. The results showed strongexpression of 161P2F10B in two of the nine colon cancer patient tissues,one of which is illustrated (FIG. 26). 161P2F10B was most stronglyexpressed on the tumor cells with a luminal cell surface but was alsoexpressed throughout all the tumor tissue.

Example 62 Detection of 161P2F10B Protein by Immunohistochemistry in aProstate Cancer Patient Specimens

Tissue specimens of prostate adenocarcinoma were obtained from eightdifferent prostate cancer patients. Frozen tissues were cut into 4micron sections and fixed in acetone for 10 minutes. The sections werethen incubated with mouse monoclonal anti-ENPP3 antibody(Coulter-Immunotech, Marseilles, France) for 3 hours. The slides werewashed three times in buffer, and further incubated with DAKO Envision+™peroxidase-conjugated goat anti-mouse secondary antibody (DAKOCorporation, Carpenteria, Calif.) for 1 hour. The sections were thenwashed in buffer, developed using the DAB kit (SIGMA Chemicals),counterstained using hematoxylin, and analyzed by bright fieldmicroscopy. The results showed expression of 161P2F10B in six of theeight prostate cancer patient tissues, one of which is illustrated (FIG.25). 161P2F10B was expressed on the tumor cells with an apparentproclivity towards the luminal cell surface.

Example 63 Detection of 161P2F10B Protein by Immunohistochemistry inNormal Tissue Specimens

Normal tissue specimens from a number of organs were obtained eitherfrom patients undergoing surgery or from autopsy. Frozen tissues werecut into 4 micron sections and fixed in acetone for 10 minutes. Thesections were then incubated with mouse monoclonal anti-ENPP3 antibody(Coulter-Immunotech, Marseilles, France) for 3 hours. The slides werewashed three times in buffer, and further incubated with DAKO Envision+™peroxidase-conjugated goat anti-mouse secondary antibody (DAKOCorporation, Carpenteria, Calif.) for 1 hour. The sections were thenwashed in buffer, developed using the DAB kit (SIGMA Chemicals),counterstained using hematoxylin, and analyzed by bright fieldmicroscopy. The results showed weak expression of 161P2F10B in some ofthe tubules in all of the kidney specimens and weak staining of someglandular epithelium in half of the prostate tissues. There was noexpression of 161P2F10B in any of the other normal tissues studiedexcept for expression in a very few cells within one lung, one bladderand two colon samples which could be mast cells (TABLE LX). As disclosedherein, Western blot and immunohistochemical analysis of kidney cancertissues and kidney cancer xenografts with mAb 97A6 showed strongextensive staining of ENPP3 in clear cell kidney carcinoma butsignificantly lower or undetectable levels in normal kidney (FIGS. 44,45, 46, and 24). Detection of 161P2F10B (ENPP3) in high grade clear cellcarcinoma and in metastatic disease.

Example 64 Detection by Immunohistochemistry of 161P2F10B ProteinExpression in Kidney Clear Cell Cancer Patient Specimens by SpecificBinding of Mouse Monoclonal Antibodies

Renal clear cell carcinoma tissue and its matched normal adjacent wereobtained from a kidney cancer patient. Frozen tissues were cut into 4micron sections and fixed in acetone for 10 minutes. The sections werethen incubated either mouse monoclonal anti-ENPP3 antibody(Coulter-Immunotech, Marseilles, France) for 3 hours (FIG. 27 panels A,D), or mouse monoclonal antibody X41(3)50 (FIG. 27 panels B, E), ormouse monoclonal antibody X41(3)37 (FIG. 27 panels C, F). The slideswere washed three times in buffer and further incubated with DAKOEnvision+™ peroxidase-conjugated goat anti-mouse secondary antibody(DAKO Corporation, Carpenteria, Calif.) for 1 hour. The sections werethen washed in buffer, developed using the DAB kit (SIGMA Chemicals),counterstained using hematoxylin, and analyzed by bright fieldmicroscopy (FIG. 27 panels A-F). The results showed strong expression of161P2F10B in the renal clear cell carcinoma patient tissue (FIG. 27panels A-C), but weakly in normal kidney (FIG. 27 panels D-F). Theexpression was predominantly around the cell periphery indicating that161P2F10B is membrane associated in kidney cancer tissues. The weakexpression detected in normal kidney was localized to the kidneyproximal tubules. As disclosed herein, Western blot andimmunohistochemical analysis of kidney cancer tissues and kidney cancerxenografts with mAb 97A6 showed strong extensive staining of ENPP3 inclear cell kidney carcinoma but significantly lower or undetectablelevels in normal kidney (FIGS. 44, 45, 46, and 24). Detection of161P2F10B (ENPP3) in high grade clear cell carcinoma and in metastaticdisease.

Example 65 Characteristics and Utility of Anti-161P2F10b MAbs

Using a variety of immunization strategies as described in Example 11, apanel of MAbs that specifically bind 161P2F10b protein was generated.The characteristics of this panel is summarized in FIG. 39 Theseantibodies specifically bind with high affinity to 161P2F10b on thesurface of endogenously-expressing and recombinant cell lines asdetermined by flow cytometry (FIGS. 28 and 40). Upon engagement ofsurface 161P2F10b, these MAbs mediate internalization of the MAb-proteincomplex (FIGS. 33, 34, and 35). These MAbs are thus useful as a specifictargeting modality for toxin-conjugates, as exemplified by the growthinhibition and induction of apoptosis of Caki-161P2F10b cells by MAbX41.50 with a saporin toxin-conjugated secondary Ab (FIG. 36). Treatmentof 161P2F10-expressing cancerous cells with the naked MAb also has atherapeutic effect in vivo as exemplified by the inhibition of UGK3tumor formation in SCID mice injected with MAb X41.50 (FIG. 23).

161P2F10b encodes phosphodiesterase enzymatic activity that is easilymonitored both in recombinant purified protein (FIG. 31) and on cells(FIG. 32). The relevance of the enzymatic activity to the function of161P2F10b may be monitored by utilization of mutants that disrupt thisactivity (FIG. 30). Engagement of 161P2F10b with MAbs may alter,disrupt, block, or downregulate 161P2F10 enzymatic activity, which mayserve as a potential therapeutic mechanism for targeting161P2F10b-expressing cancers and diseased tissues. Engagement of cellsurface 161P2F10b cells with a subset of the MAbs listed in FIG. 39 doesmediate internalization and marked downregulation of cell surfaceenzymatic activity (FIGS. 37 and 38) thus demonstrating the utility ofthe MAbs for disrupting the function of 161P2F10b in cells and tissues.

161P2F10b protein and the MAbs that bind it are useful in the diagnosisof 161P2F10b-expressing cancer and diseased tissues Immunohistochemicalanalysis of the panel of MAbs, as summarized in FIG. 39, specificallystain (to varying degrees) a variety of kidney cancer samples withlittle to no staining of adjacent normal tissues. These MAbs are thususeful as diagnostic reagents for a variety of 161P2F10b-expressingcancers by immunohistochemistry and are potentially useful as imagingreagents in patients. In addition, the MAbs were used (specificallyX48.54 and X41.29, but others that do not compete for the same epitopeare also used) to demonstrate the shedding and/or secretion of theprotein from 161P2F10b-expressing cancer cells and tissues (FIGS. 42 and43). This supports the utility of 161P2F10b as a serum and/or urinediagnostic marker and the MAbs as reagents to quantitatively measureserum and/or urine concentrations of 161P2F10b protein.

Throughout this application, various website data content, publications,patent applications and patents are referenced. The disclosures of eachof these references are hereby incorporated by reference herein in theirentireties.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

Tables

TABLE I Tissues that Express 161P2F10B: Exemplary Normal Tissues:Prostate, Kidney Malignant Tissues Kidney, Uterus, Pancreas, Prostate,Colon, Lung, Bone, Lymphoma, Breast, Ovary,

TABLE II Amino Acid Abbreviations SINGLE LETTER THREE LETTER FULL NAME FPhe phenylalanine L Leu leucine S Ser serine Y Tyr tyrosine C Cyscysteine W Trp tryptophan P Pro proline H His histidine Q Gln glutamineR Arg arginine I Ile isoleucine M Met methionine T Thr threonine N Asnasparagine K Lys lysine V Val valine A Ala alanine D Asp aspartic acid EGlu glutamic acid G Gly glycine

TABLE III Amino Acid Substitution Matrix A C D E F G H I K L M N P Q R ST V W Y . 4 0 −2 −1 −2 0 −2 −1 −1 −1 −1 −2 −1 −1 −1 1 0 0 −3 −2 A 9 −3−4 −2 −3 −3 −1 −3 −1 −1 −3 −3 −3 −3 −1 −1 −1 −2 −2 C 6 2 −3 −1 −1 −3 −1−4 −3 1 −1 0 −2 0 −1 −3 −4 −3 D 5 −3 −2 0 −3 1 −3 −2 0 −1 2 0 0 −1 −2 −3−2 E 6 −3 −1 0 −3 0 0 −3 −4 −3 −3 −2 −2 −1 1 3 F 6 −2 −4 −2 −4 −3 0 −2−2 −2 0 −2 −3 −2 −3 G 8 −3 −1 −3 −2 1 −2 0 0 −1 −2 −3 −2 2 H 4 −3 2 1 −3−3 −3 −3 −2 −1 3 −3 −1 I 5 −2 −1 0 −1 1 2 0 −1 −2 −3 −2 K 4 2 −3 −3 −2−2 −2 −1 1 −2 −1 L 5 −2 −2 0 −1 −1 −1 1 −1 −1 M 6 −2 0 0 1 0 −3 −4 −2 N7 −1 −2 −1 −1 −2 −4 −3 P 5 1 0 −1 −2 −2 −1 Q 5 −1 −1 −3 −3 −2 R 4 1 −2−3 −2 S 5 0 −2 −2 T 4 −3 −1 V 11 2 W 7 Y Adapted from the GCG Software9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix).The higher the value, the more likely a substitution is found inrelated, natural proteins.

TABLE IV (A) HLA Class I Supermotifs/Motifs POSITION POSITION POSITION CTerminus 2 (Primary 3 (Primary (Primary Anchor) Anchor) Anchor)SUPERMOTIF A1 TI LVMS FWY A2 LIVM ATQ IV MATL A3 VSMA TLI RK A24 YFWIVLMT FI YWLM B7 P VILF MWYA B27 RHK FYL WMIVA B44 E D FWYLIMVA B58 ATSFWY LIVMA B62 QL IVMP FWY MIVLA MOTIFS A1 TSM Y A1 DE AS Y A2.1 LM VQIATV LIMAT A3 LMVISATF CGD KYR HFA A11 VTMLISAGN CDF K RYH A24 YF WM FLIWA*3101 MVT ALIS R K A*3301 MVALF IST RK A*6801 AVT MSLI RK B*0702 P LMFWYAIV B*3501 P LMFWY IVA B51 P LIVF WYAM B*5301 P IMFWY ALV B*5401 PATIV LMFWY Bolded residues are preferred, italicized residues are lesspreferred: A peptide is considered motif-bearing if it has primaryanchors at each primary anchor position for a motif or supermotif asspecified in the above table.

TABLE IV (B) HLA Class II Supermotif 1 6 9 W, F, Y, V, .I, L A, V, I, L,P, C, S, T A, V, I, L, C, S, T, M, Y

TABLE IV (C) HLA Class II Motifs MOTIFS 1° anchor 1 2 3 4 5 1° anchor 67 8 9 DR4 preferred FMYLIVW M T I VSTCPALIM MH MH deleterious W R WDEDR1 preferred MFLIVWY PAMQ VMATSPLIC M AVM deleterious C CH FD CWD GDE DDR7 preferred MFLIVWY M W A IVMSACTPL M IV deleterious C G GRD N G DR3MOTIFS 1° anchor 1 2 3 1° anchor 4 5 1° anchor 6 Motif a preferredLIVMFY D Motif b preferred LIVMFAY DNQEST KRH DR Supermotif MFLIVWYVMSTACPLI Italicized residues indicate less preferred or “tolerated”residues

TABLE IV (D) HLA Class I Supermotifs SUPER- POSITION: MOTIFS 1 2 3 4 5 67 8 C-terminus A1 1° Anchor 1° Anchor TILVMS FWY A2 1° Anchor 1° AnchorLIVMATQ LIVMAT A3 Preferred 1° Anchor YFW YFW YFW P 1° Anchor VSMATLI(4/5) (3/5) (4/5) (4/5) RK deleterious DE (3/5); DE P (5/5) (4/5) A24 1°Anchor 1° Anchor YFWIVLMT FIYWLM B7 Preferred FWY (5/5) 1° Anchor FWYFWY 1° Anchor LIVM (3/5) P (4/5) (3/5) VILFMWYA deleterious DE (3/5); DEG QN DE P(5/5); (3/5) (4/5) (4/5) (4/5) G(4/5); A(3/5); QN(3/5) B27 1°Anchor 1° Anchor RHK FYLWMIVA B44 1° Anchor 1° Anchor ED FWYLIMVA B58 1°Anchor 1° Anchor ATS FWYLIVMA B62 1° Anchor 1° Anchor QLIVMP FWYMIVLAItalicized residues indicate less preferred or “tolerated” residues

TABLE IV (E) HLA Class I Motifs POSITION 9 or C- C- 1 2 3 4 5 6 7 8terminus terminus A1 preferred GFYW 1° Anchor DEA YFW P DEQN YFW 1°Anchor 9-mer STM Y deleterious DE RHKLIVMP A G A A1 preferred GRHKASTCLIVM 1° Anchor GSTC ASTC LIVM DE 1° Anchor 9-mer DEAS Y deleteriousA RHKDEPYFW DE PQN RHK PG GP A1 preferred YFW 1° Anchor DEAQN A YFWQNPASTC GDE P 1° Anchor 10-mer STM Y deleterious GP RHKGLIVM DE RHK QNARHKYFW RHK A A1 preferred YFW STCLIVM 1° Anchor A YFW PG G YFW 1° Anchor10-mer DEAS Y deleterious RHK RHKDEPYFW P G PRHK QN A2.1 preferred YFW1° Anchor YFW STC YFW A P 1° Anchor 9-mer LMIVQAT VLIMAT deleterious DEPDERKH RKH DERKH POSITION: C- 1 2 3 4 5 6 7 8 9 Terminus A2.1 preferredAYFW 1° Anchor LVIM G G FYWL 1° Anchor 10-mer LMIVQAT VIM VLIMATdeleterious DEP DE RKHA P RKH DERKH RKH A3 preferred RHK 1° Anchor YFWPRHKYF A YFW P 1° Anchor LMVISA W KYRHFA TFCGD deleterious DEP DE A11preferred A 1° Anchor YFW YFW A YFW YFW P 1° Anchor VTLMISA KRYH GNCDFdeleterious DEP A G A24 preferred YFWRHK 1° Anchor STC YFW YFW 1° Anchor9-mer YFWM FLIW deleterious DEG DE G QNP DERHK G AQN A24 Preferred 1°Anchor P YFWP P 1° Anchor 10-mer YFWM FLIW Deleterious GDE QN RHK DE AQN DEA A3101 Preferred RHK 1° Anchor YFW P YFW YFW AP 1° Anchor MVTALISRK Deleterious DEP DE ADE DE DE DE A3301 Preferred 1° Anchor YFW AYFW 1°Anchor MVALFIST RK Deleterious GP DE A6801 Preferred YFWSTC 1° AnchorYFWLIV YFW P 1° Anchor AVTMSLI M RK deleterious GP DEG RHK A B0702Preferred RHKFWY 1° Anchor RHK RHK RHK RHK PA 1° Anchor P LMFWYAI VPOSITION 9 or C- C- 1 2 3 4 5 6 7 8 terminus terminus A1 preferred GFYW1° Anchor DEA YFW P DEQN YFW 1° Anchor 9-mer STM Y deleterious DERHKLIVMP A G A A1 preferred GRHK ASTCLIVM 1° Anchor GSTC ASTC LIVM DE 1°Anchor 9-mer DEAS Y deleterious A RHKDEPYFW DE PQN RHK PG GP deleteriousDEQNP DEP DE DE GDE QN DE B3501 Preferred FWYLIVM 1° Anchor FWY FWY 1°Anchor P LMFWYIV A deleterious AGP G G B51 Preferred LIVMFWY 1° AnchorFWY STC FWY G FWY 1° Anchor P LIVFWYA M deleterious AGPDER DE G DEQN GDEHKSTC B5301 preferred LIVMFWY 1° Anchor FWY STC FWY LIVMFWY FWY 1°Anchor P IMFWYAL V deleterious AGPQN G RHKQN DE B5401 preferred FWY 1°Anchor FWYLIVM LIVM ALIVM FWYA 1° Anchor P P ATIVLMF WY deleteriousGPQNDE GDESTC RHKDE DE QNDGE DE

TABLE IV (F) Summary of HLA-supertypes Overall phenotypic frequencies ofHLA-supertypes in different ethnic populations Specificity Phenotypicfrequency Supertype Position 2 C-Terminus Caucasian N.A. Black JapaneseChinese Hispanic Average B7 P AILMVFWY 43.2 55.1 57.1 43.0 49.3 49.5 A3AILMVST RK 37.5 42.1 45.8 52.7 43.1 44.2 A2 AILMVT AILMVT 45.8 39.0 42.445.9 43.0 42.2 A24 YF (WIVLMT) FI (YWLM) 23.9 38.9 58.6 40.1 38.3 40.0B44 E (D) FWYLIMVA 43.0 21.2 42.9 39.1 39.0 37.0 A1 TI (LVMS) FWY 47.116.1 21.8 14.7 26.3 25.2 B27 RHK FYL (WMI) 28.4 26.1 13.3 13.9 35.3 23.4B62 QL (IVMP) FWY (MIV) 12.6 4.8 36.5 25.4 11.1 18.1 B58 ATS FWY (LIV)10.0 25.1 1.6 9.0 5.9 10.3

TABLE IV (G) Calculated population coverage afforded by differentHLA-supertype combinations Phenotypic frequency HLA-supertypes CaucasianN.A Blacks Japanese Chinese Hispanic Average A2, A3 and B7 83.0 86.187.5 88.4 86.3 86.2 A2, A3, B7, A24, 99.5 98.1 100.0 99.5 99.4 99.3 B44and A1 A2, A3, B7, A24, 99.9 99.6 100.0 99.8 99.9 99.8 B44, A1, B27,B62, and B58 Motifs indicate the residues defining supertypespecificites. The motifs incorporate residues determined on the basis ofpublished data to be recognized by multiple alleles within thesupertype. Residues within brackets are additional residues alsopredicted to be tolerated by multiple alleles within the supertype.

TABLE V Frequently Occurring Motifs avrg. % Name identity DescriptionPotential Function zf-C2H2 34% Zinc finger, C2H2 type Nucleicacid-binding protein functions as transcription factor, nuclear locationprobable cytochrome_b_N 68% Cytochrome b(N- membrane bound oxidase,generate terminal)/b6/petB superoxide Ig 19% Immunoglobulin domaindomains are one hundred amino acids long and include a conservedintradomain disulfide bond. WD40 18% WD domain, G-beta repeat tandemrepeats of about 40 residues, each containing a Trp-Asp motif. Functionin signal transduction and protein interaction PDZ 23% PDZ domain mayfunction in targeting signaling molecules to sub-membranous sites LRR28% Leucine Rich Repeat short sequence motifs involved inprotein-protein interactions Pkinase 23% Protein kinase domain conservedcatalytic core common to both serine/threonine and tyrosine proteinkinases containing an ATP binding site and a catalytic site PH 16% PHdomain pleckstrin homology involved in intracellular signaling or asconstituents of the cytoskeleton EGF 34% EGF-like domain 30-40amino-acid long found in the extracellular domain of membrane- boundproteins or in secreted proteins Rvt 49% Reverse transcriptase(RNA-dependent DNA polymerase) Ank 25% Ank repeat Cytoplasmic protein,associates integral membrane proteins to the cytoskeleton Oxidored_q132% NADH-Ubiquinone/plastoquinone membrane associated. Involved in(complex I), various chains proton translocation across the membraneEfhand 24% EF hand calcium-binding domain, consists of a12 residue loopflanked on both sides by a 12 residue alpha-helical domain Rvp 79%Retroviral aspartyl Aspartyl or acid proteases, centered on protease acatalytic aspartyl residue Collagen 42% Collagen triple helix repeatextracellular structural proteins involved (20 copies) in formation ofconnective tissue. The sequence consists of the G-X-Y and thepolypeptide chains forms a triple helix. Fn3 20% Fibronectin type IIIdomain Located in the extracellular ligand- binding region of receptorsand is about 200 amino acid residues long with two pairs of cysteinesinvolved in disulfide bonds 7tm_1 19% 7 transmembrane receptor sevenhydrophobic transmembrane (rhodopsin family) regions, with theN-terminus located extracellularly while the C-terminus is cytoplasmic.Signal through G proteins

TABLE VI Motifs and Post-translational Modifications of 161P2F108N-glycosylation site: Number of matches: 10 1 236-239 NFSL (SEQ ID NO:41) 2 279-282 NGSF (SEQ ID NO: 42) 3 290-293 NGSV (SEQ ID NO: 43) 4426-429 NLSC (SEQ ID NO: 44) 5 533-536 NGTH (SEQ ID NO: 45) 6 582-585NSTQ (SEQ ID NO: 46) 7 594-597 NLTQ (SEQ ID NO: 47) 8 687-690 NITH (SEQID NO: 48) 9 699-702 NRTS (SEQ ID NO: 49) 10 789-792 NKSH (SEQ ID NO:50) cAMP- and cGMP-dependent protein kinase phosphorylation site 14-17KKNT (SEQ ID NO: 51) Protein kinase C phosphorylation site Number ofmatches: 13 1 17-19 TLK 2 53-55 SCR 3 428-430 SCR 4 62-64 SFR 5 92-94STR 6 240-242 SSK 7 335-337 SAR 8 53-55 SCR 9 428-430 SCR 10 502-5O4 SFK11 603-605 TVK 12 676-678 SQK 13 698-700 SNR Casein kinase IIphosphorylation site Number of matches: 15 1 88-91 TCVE (SEQ ID NO: 52)2 106-109 TRLE (SEQ ID NO: 53) 3 114-117 SCSD (SEQ ID NO: 54) 4 138-141SWLE (SEQ ID NO: 55) 5 240-243 SSKE (SEQ ID NO: 56) 6 502-505 SFKE (SEQID NO: 57) 7 507-510 TEVE (SEQ ID NO: 58) 8 551-554 SHAE (SEQ ID NO: 59)9 584-587 TQLE (SEQ ID NO: 60) 10 596-599 TQEE (SEQ ID NO: 61) 11660-663 TVPD (SEQ ID NO: 62) 12 704-707 SQYD (SEQ ID NO: 63) 13 813-816TNVE (SEQ ID NO: 64) 14 817-820 SCPE (SEQ ID NO: 65) 15 846-849 TGLD(SEQ ID NO: 66) Tyrosine kinase phosphorylation site 700-706 RTSDSQY(SEQ ID NO: 67) N-myristoylation site Number of matches: 11 1 38-43GLGLGL (SEQ ID NO: 68) 2 40-45 GLGLGL (SEQ ID NO: 69) 3 38-43 GLGLGL(SEQ ID NO: 70) 4 40-45 GLGLGL (SEQ ID NO: 71) 5 65-70 GLENCR (SEQ IDNO: 72) 6 222-227 GIIDNN (SEQ ID NO: 73) 7 263-268 GLKAAT (SEQ ID NO:74) 8 273-278 GSEVAI (SEQ ID NO: 75) 9 280-285 GSFPSI (SEQ ID NO: 76) 10331-336 GGPVSA (SEQ ID NO: 77) 11 374-379 GMDQTY (SEQ ID NO: 78) Cellattachment sequence 78-80 RGD Somatomedin B domain signature Number ofmatches: 2 1 69-89 CRCDVACKDRGDCCWDFEDTC (SEQ ID NO: 79) 2 113-133CSCSDDCLQKKDCCADYKSVC (SEQ ID NO: 80)

TABLE VII Search Peptides 161P2F10B variant 1 (SEQ ID NO: 81)   1MESTLTLATE QPVKKNTLKK YKIACIVLLA LLVIMSLGLG LGLGLRKLEK QGSCRKKCFD  61ASFRGLENCR CDVACKDRGD CCWDFEDTCV ESTRIWMCNK FRCGETRLEA SLCSCSDDCL 121QKKDCCADYK SVCQGETSWL EENCDTAQQS QCPEGFDLPP VILFSMDGFR AEYLYTWDTL 181MPNINKLKTC GIHSKYMRAM YPTKTFPNHY TIVTGLYPES HGIIDNNMYD VNLNKNFSLS 241SKEQNNPAWW HGQPMWLTAM YQGLKAATYF WPGSEVAING SFPSIYMPYN GSVPFEERIS 301TLLKWLDLPK AERPRFYTMY FEEPDSSGHA GGPVSARVIK ALQVVDHAFG MLMEGLKQRN 361LHNCVNIILL ADHGMDQTYC NKMEYMTDYF PRINFFYMYE GPAPRIRAHN IPHDFFSFNS 421EEIVRNLSCR KPDQHFKPYL TPDLPKRLHY AKNVRIDKVE LFVDQQWLAV RSKSNTNCGG 481GNHGYNNEFR SMEAIFLAHG PSFKEKTEVE PFENIEVYNL MCDLLRIQPA PNNGTHGSLN 541HLLKVPFYEP SHAEEVSKFS VCGFANPLPT ESLDCFCPHL QNSTQLEQVN QMLNLTQEEI 601TATVKVNLPF GRPRVLQKNV DHCLLYHREY VSGFGKAMRM PMWSSYTVPQ LGDTSPLPPT 661VPDCLRADVR VPPSESQKCS FYLADKNITH GFLYPPASNR TSDSQYDALI TSNLVPMYEE 721FRKMWDYFHS VLLIKHATER NGVNVVSGPI FDYNYDGHFD APDEITKHLA NTDVPIPTHY 781FVVLTSCKNK SHTPENCPGW LDVLPFIIPH RPTNVESCPE GKPEALWVEE RFTAHIARVR 841DVELLTGLDF YQDKVQPVSE ILQLKTYLPT FETTI Variant 2 9-mers SCSDDCLQ

KDCCADYK (SEQ ID NO: 82) 10-mers CSCSDDCLQ

KDCCADYKS (SEQ ID NO: 83) 15-mers LEASLCSCSDDCLQ

KDCCADYKSVCQGE (SEQ ID NO: 84) Variant 3 9-mers PTNVESCP

GKPEALWV (SEQ ID NO: 85) 10-mers RPTNVESCP

GKPEALWVE (SEQ ID NO: 86) 15-mers FIIPHRPTNVESCP

GKPEALWVEERFTA (SEQ ID NO: 87) Variant 4 9-mers TYLPTFET

I (SEQ ID NO: 88) 10-mers KTYLPTFET

I (SEQ ID NO: 89) 15-mers EILQLKTYLPTFET

I (SEQ ID NO: 90)

TABLE VIII V1-HLA-A1-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 3; eachstart position is specified, the length of peptideis 9 amino acids, and the end position for eachpeptide is the start position plus eight. Start Subsequence Score 105SMDGFRAEY 25.000 754 NVESCPEGK 18.000  55 CSDDCLQKK 15.000 446 KTEVEPFEN11.250 245 WLDLPKAER 10.000 798 VSEILQLKT 6.750 317 QTYCNKMEY 6.250 371KPDQHFKPY 6.250  10 RCDVACKDR 5.000 314 GMDQTYCNK 5.000 322 KMEYMTDYF4.500 454 NIEVYNLMC 4.500  29 CVESTRIWM 4.500 650 ITSNLVPMY 2.500 118DTLMPNINK 2.500 685 VVSGPIFDY 2.500 559 NVDHCLLYH 2.500 711 NTDVPIPTH2.500 740 WLDVLPFII 2.500 402 FVDQQWLAV 2.500 431 SMEAIFLAH 2.250 610RVPPSESQK 2.000 379 YLTPDLPKR 2.000 613 PSESQKCSF 1.350 213 GSEVAINGS1.350 359 NSEEIVRNL 1.350 128 KTCGIHSKY 1.250 326 MTDYFPRIN 1.250 426NNEFRSMEA 1.125 632 FLYPPASNR 1.000 163 IIDNNMYDV 1.000 512 SLDCFCPHL1.000  66 CADYKSVCQ 1.000 653 NLVPMYEEF 1.000 606 RADVRVPPS 1.000  54SCSDDCLQK 1.000 767 WVEERFTAH 0.900 448 EVEPFENIE 0.900 525 QLEQVNQML0.900  79 WLEENCDTA 0.900 492 HAEEVSKFS 0.900 537 QEEITATVK 0.900   5GLENCRCDV 0.900  47 RLEASLCSC 0.900 641 TSDSQYDAL 0.750 626 KNITHGFLY0.625 558 KNVDHCLLY 0.625 783 ELLTGLDFY 0.500  62 KKDCCADYK 0.500 310LADHGMDQT 0.500 167 NMYDVNLNK 0.500 562 HCLLYHREY 0.500 197 LTAMYQGLK0.500 699 FDAPDEITK 0.500 787 GLDFYQDKV 0.500   83 NCDTAQQSQ 0.500 593DTSPLPPTV 0.500 284 VVDHAFGML 0.500 144 KTFPNHYTI 0.500 417 NCGGGNHGY0.500 350 NIPHDFFSF 0.500  97 DLPPVILFS 0.500 742 DVLPFIIPH 0.500 250KAERPRFYT 0.450 292 LMEGLKQRN 0.450 677 ATERNGVNV 0.450 261 FEEPDSSGH0.450 618 KCSFYLADK 0.400 571 VSGFGKAMR 0.300 536 TQEEITATV 0.270 193QPMWLTAMY 0.250 687 SGPIFDYNY 0.250 772 FTAHIARVR 0.250 655 VPMYEEFRK0.250 476 HGSLNHLLK 0.250 712 TDVPIPTHY 0.250 550 FGRPRVLQK 0.250 601VPDCLRADV 0.250 578 MRMPMWSSY 0.250 546 VNLPFGRPR 0.250 461 MCDLLRIQP0.250 395 RIDKVHLFV 0.250 586 YTVPQLGDT 0.250  96 FDLPPVILF 0.250 509PTESLDCFC 0.225 758 CPEGKPEAL 0.225 733 TPENCPGWL 0.225 493 AEEVSKFSV0.225  43 CGETRLEAS 0.225 722 VVLTSCKNK 0.200 746 FIIPHRPTN 0.200 436FLAHGPSFK 0.200 743 VLPFIIPHR 0.200 133 HSKYMRAMY 0.150 201 YQGLKAATY0.150 231 GSVPFEERI 0.150 686 VSGPIFDYN 0.150 382 PDLPKRLHY 0.125 191HGQPMWLTA 0.125 701 APDEITKHL 0.125 V2-HLA-A1-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 5; eachstart position is specified, the length of peptideis 9 amino acids, and the end position for eachpeptide is the start position plus eight. Start Subsequence Score 2CSDDCLQRK 15.000 9 RKDCCADYK 0.500 1 SCSDDCLQR 0.500 5 DCLQRKDCC 0.010 8QRKDCCADY 0.005 3 SDDCLQRKD 0.003 6 CLQRKDCCA 0.001 4 DDCLQRKDC 0.001 7LQRKDCCAD 0.000 V3-HLA-A1-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 7; eachstart position is specified, the length of peptideis 9 amino acids, and the end position for eachpeptide is the start position plus eight. Start Subsequence Score 3NVESCPGGK 18 6 SCPGGKPEA 0.02 5 ESCPGGKPE 0.015 9 GGKPEALWV 0.013 2TNVESCPGG 0.005 7 CPGGKPEAL 0.003 1 PTNVESCPG 0.003 4 VESCPGGKP 0 8PGGKPEALW 0 V4-HLA-A1-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 9; eachstart position is specified, the length of peptideis 9 amino acids, and the end position for eachpeptide is the start position plus eight. Start Subsequence Score 2YLPTFETPI 0.01 1 TYLPTFETP 0.001

TABLE IX Start Subsequence Score V1-HLA-A1-10mers-161P2F10BEach peptide is a portion of SEQ ID NO: 3; each start positionis specified, the length of peptide is 10 amino acids, and the endposition for each peptide is the start position plus nine. 798VSEILQLKTY 67.500 711 NTDVPIPTHY 62.500 326 MTDYFPRINF 62.500 781DVELLTGLDF 45.000 448 EVEPFENIEV 45.000 220 FSFPSIYMPY 37.500 381TPDLPKRLHY 31.250 310 LADHGMDQTY 25.000 686 VSGPIFDYNY 15.000 213GSEVAINGSF 13.500 402 FVDQQWLAVR 10.000 559 NVDHCLLYHR 10.000 613PSESQKCSFY 6.750 525 QLEQVNQMLN 4.500 492 HAEEVSKFSV 4.500 536TQEEITATVK 2.700 684 NVVSGPIFDY 2.500 698 HFDAPDEITK 2.500 284VVDHAFGMLM 2.500 658 YEEFRKMWDY 2.250 446 KTEVEPFENI 2.250 762KPEALWVEER 2.250 742 DVLPFIIPHR 2.000 119 TLMPNINKLK 2.000 767WVEERFTAHI 1.800 53 CSCSDDCLQK 1.500 641 TSDSQYDALI 1.500 104 FSMDGFRAEY1.500 359 NSEEIVRNLS 1.350 349 HNIPHDFFSF 1.250 328 DYFPRINFFY 1.250 601VPDCLRADVR 1.250 95 GFDLPPVILF 1.250 157 YPESHGIIDN 1.125 654 LVPMYEEFRK1.000 653 NLVPMYEEFR 1.000 649 LITSNLVPMY 1.000 47 RLEASLCSCS 0.900 29CVESTRIWMC 0.900 5 GLENCRCDVA 0.900 110 RAEYLYTWDT 0.900 79 WLEENCDTAQ0.900 431 SMEAIFLAHG 0.900 356 FSFNSEEIVR 0.750 785 LTGLDFYQDK 0.500 623LADKNITHGF 0.500 83 NCDTAQQSQC 0.500 66 CADYKSVCQG 0.500 541 TATVKVNLPF0.500 10 RCDVACKDRG 0.500 794 KVQPVSEILQ 0.500 787 GLDFYQDKVQ 0.500 163IIDNNMYDVN 0.500 512 SLDCFCPHLQ 0.500 461 MCDLLRIQPA 0.500 217AINGSFPSIY 0.500 97 DLPPVILFSM 0.500 292 LMEGLKQRNL 0.450 487 FYEPSHAEEV0.450 92 CPEGFDLPPV 0.450  43 CGETRLEASL 0.450 235 FEERISTLLK 0.450 454NIEVYNLMCD 0.450 261 FEEPDSSGHA 0.450 758 CPEGKPEALW 0.450 338MYEGPAPRIR 0.450 316 DQTYCNKMEY 0.375 756 ESCPEGKPEA 0.300 263EPDSSGHAGG 0.250 740 WLDVLPFIIP 0.250 586 YTVPQLGDTS 0.250 314GMDQTYCNKM 0.250 479 LNHLLKVPFY 0.250 139 AMYPTKTFPN 0.250 168MYDVNLNKNF 0.250 25 FEDTCVESTR 0.250 377 KPYLTPDLPK 0.250 105 SMDGFRAEYL0.250 591 LGDTSPLPPT 0.250 166 NNMYDVNLNK 0.250 144 KTFPNHYTIV 0.250 371KPDQHFKPYL 0.250 690 IFDYNYDGHF 0.250 426 NNEFRSMEAI 0.225 74 QGETSWLEEN0.225 360 SEEIVRNLSC 0.225 677 ATERNGVNVV 0.225 570 YVSGFGKAMR 0.200 129TCGIHSKYMR 0.200 721 FVVLTSCKNK 0.200 682 GVNVVSGPIF 0.200 196WLTAMYQGLK 0.200 434 AIFLAHGPSF 0.200 54S CSDDCLQKK 0.200 478 SLNHLLKVPF0.200 437 LAHGPSFKEK 0.200 250 KAERPRFYTM 0.180 231 GSVPFEERIS 0.150 192GQPMWLTAMY 0.150 V2-HLA-A1-10mers-161P2F10B EacH peptide is a portion ofSEQ ID nO: 5; each start position is specified, the length of peptideis 10 amino acids, and the end position for each peptide is thestart position plus nine. 1 CSCSDDCLQR 0.750 2 SCSDDCLQRK 0.200 3CSDDCLQRKD 0.075 10 RKDCCADYKS 0.050 4 SDDCLQRKDC 0.025 6 DCLQRKDCCA0.010 8 LQRKDCCADY 0.002 9 QRKDCCADYK 0.001 5 DDCLQRKDCC 0.001 7CLQRKDCCAD 0.000 V3-HLA-A1-10mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 7; each start position is specified, the length of peptideis 10 amino acids, and the end position for each peptide is thestart position plus nine. 6 ESCPGGKPEA 0.3 4 NVESCPGGKP 0.09 3TNVESCPGGK 0.05 7 SCPGGKPEAL 0.01 2 PTNVESCPGG 0.005 8 CPGGKPEALW 0.0051 RPTNVESCPG 0.003 10 GGKPEALWVE 0 9 PGGKPEALWV 0 5 VESCPGGKPE 0V4-HLA-A1-10mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 9; each start position is specified, the length of peptideis 10 amino acids, and the end position for each peptide is thestart position plus nine. 2 TYLPTFETPI 0.005 1 KTYLPTFETP 0.003

TABLE X Start Subsequence Score V1-HLA-A0201-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 3; each start positionis specified, the length of peptide is 9 amino acids, and the endposition for each peptide is the start position plus eight. 663KMWDYFHSV 11367.476 563 CLLYHREYV 693.538 325 YMTDYFPRI 270.002 807YLPTFETTI 182.365 119 TLMPNINKL 181.794 196 WLTAMYQGL 147.401 459NLMCDLLRI 88.783 113 YLYTWDTLM 73.129 547 NLPFGRPRV 69.552 765 ALWVEERFT68.037 740 WLDVLPFII 45.649 238 RISTLLKWL 37.157 155 GLYPESHGI 33.385512 SLDCFCPHL 32.471 579 TMPMWSSYT 29.601 199 AMYQGLKAA 26.408 395RIDKVHLFV 21.039 402 FVDQQWLAV 19.036 524 TQLEQVNQM 17.575 747 IIPHRPTNV16.258 163 IIDNNMYDV 14.957 400 HLFVDQQWL 14.781 787 GLDFYQDKV 13.632 90SQCPEGFDL 12.562 693 YNYDGHFDA 11.352 283 QVVDHAFGM 10.337 300 NLHNCVNII9.838 555 VLQKNVDHC 9.518 532 MLNLTQEEI 8.691 570 YVSGFGKAM 7.599 802LQLKTYLPT 7.129 500 SVCGFANPL 7.103 805 KTYLPTFET 6.723 430 RSMEAIFLA6.563 277 RVIKALQVV 5.739 171 VNLNKNFSL 5.087 59 CLQKKDCCA 4.968 534NLTQEEITA 4.968 383 DLPKRLHYA 4.713 800 EILQLKTYL 4.483 5 GLENCRCDV4.451 452 FENIEVYNL 4.395 307 IILLADHGM 4.297 714 VPIPTHYFV 4.245 477GSLNHLLKV 3.864 111 AEYLYTWDT 3.478 488 YEPSHAEEV 3.048 79 WLEENCDTA2.938 580 MPMWSSYTV 2.856 30 VESTRIWMC 2.833 217 AINGSFPSI 2.726 649LITSNLVPM 2.671 51 SLCSCSDDC 2.434 670 SVLLIKHAT 2.413 449 VEPFENIEV2.299 380 LTPDLPKRL 2.068 21 CCWDFEDTC 2.O55 144 KTFPNHYTI 1.876 297KQRNLHNCV 1.876 240 STLLKWLDL 1.866 536 TQEEITATV 1.850 535 LTQEEITAT1.659 356 FSFNSEEIV 1.552 528 QVNQMLNLT 1.500 583 WSSYTVPQL 1.475 622YLADKNITH 1.405 525 QLEQVNQML 1.367 794 KVQPVSEIL 1.314 72 VCQGETSWL1.304 467 IQPAPNNGT 1.284 233 VPFEERIST 1.255 192 GQPMWLTAM 1.159 456EVYNLMCDL 1.032 131 GIHSKYMRA 1.025 280 KALQVVDHA 1.007 291 MLMEGLKQR0.884 427 NEFRSMEAI 0.846 784 LLTGLDFYQ 0.808 447 TEVEPEENI 0.774 715PIPTHYFVV 0.750 250 KAERPRFYT 0.740 98 LPPVILFSM 0.735 47 RLEASLCSC0.731 330 FPRINFEYM 0.687 474 GTHGSLNHL 0.682 337 YMYEGPAPR 0.650 274VSARVIKAL 0.545 521 QNSTQLEQV 0.512 540 ITATVKVNL 0.504 493 AEEVSKFSV0.502 270 AGGPVSARV 0.454 665 WDYFHSVLL 0.437 790 FYQDKVQPV 0.419 44GETRLEASL 0.415 190 WHGQPMWLT 0.411 656 PMYEEFRKM 0.394 436 FLAHGPSFK0.377 527 EQVNQMLNL 0.374 115 YTWDTLMPN 0.373 708 HLANTDVPI 0.355V2-A0201-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 5; each start position is specified, the length of peptideis 9 amino acids, and the end position for each peptide is thestart position plus eight. 6 CLQRKDCCA 4.968 5 DCLQRKDCC 0.004 4DDCLQRKDC 0.001 1 SCSDDCLQR 0.000 2 CSDDCLQRK 0.000 7 LQRKDCCAD 0.000 9RKDCCADYK 0.000 3 SDDCLQRKD 0.000 8 QRKDCCADY 0.000V3-HLA-A2-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 7; each start position is specified, the length of peptideis 9 amino acids, and the end position for each peptide is thestart position plus eight. 9 GGKPEALWV 0.087 7 CPGGKPEAL 0.068 6SCPGGKPEA 0.032 2 TNVESCPGG 0.002 4 VESCPGGKP 0 1 PTNVESCPG 0 3NVESCPGGK 0 8 PGGKPEALW 0 5 ESCPGGKPE 0 V4-HLA-A2-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 9 each start positionis specified, the length of peptide is 9 amino acids, and the endposition for each peptide is the start position plus eight. 2 YLPTFETPI182.365 1 TYLPTFETP 0

TABLE XI Start Subsequence Score V1-HLA-A0201-10mers-161P2F10BEach peptide is a portion of SEQ ID NO: 3; each start positionis specified, the length of peptide is 10 amino acids, and the endposition amino for each peptide is the start position plus nine. 663KMWDYFHSVL 2862.980 337 YMYEGPAPRI 454.740 765 ALWVEERFTA 239.160 102ILFSMDGFRA 181.243 632 FLYPPASNRT 109.693 379 YLTPDLPKRL 98.267 162GIIDNNMYDV 90.183 309 LLADHGMDQT 58.537 115 YTWDTLMPNI 52.169 579RMPMWSSYTV 50.232 746 FIIPHRPTNV 43.992 555 VLQKNVDHCL 36.316 407WLAVRSKSNT 34.279 34 RIWMCNKFRC 32.884 524 TQLEQVNQML 32.857 600TVPDCLRADV 24.952 801 ILQLKTYLPT 19.003 199 AMYQGLKAAT 17.222 534NLTQEEITAT 17.140 105 SMDGFRAEYL 16.632 21 CCWDFEDTCV 15.450 531QMLNLTQEEI 13.661 520 LQNSTQLEQV 13.511 614 SESQKCSFYL 13.251 648ALITSNLVPM 11.426 51 SLCSCSDDCL 10.468 387 RLHYAKNVRI 10.433 71SVCQGETSWL 10.281 120 LMPNINKLKT 9.149 300 NLHNCVNIIL 8.759 795VQPVSEILQL 8.469 233 VPFEERISTL 8.271 144 KTFPNHYTIV 7.693 4 RGLENCRCDV6.887 535 LTQEEITATV 6.733 97 DLPPVILFSM 4.970 282 LQVVDHAFGM 4.966 400HLFVDQQWLA 4.687 767 WVEERFTAHI 4.187 371 KPDQHFKPYL 4.080 224SIYMPYNGSV 3.978 786 TGLDFYQDKV 3.375 622 YLADKNITHG 3.233 207ATYFWPGSEV 3.091 546 VNLPFGRPRV 2.856 714 VPIPTHYFVV 2.753 458YNLMCDLLRI 2.666 532 MLNLTQEEIT 2.545 713 DVPIPTHYFV 2.510 499FSVCGFANPL 2.438 799 SEILQLKTYL 2.285 291 MLMEGLKQRN 1.922 283QVVDHAFGML 1.893 136 YMRAMYPTKT 1.882 554 RVLQKNVDHC 1.813 155GLYPESHGII 1.779 547 NLPFGRPRVL 1.752 455 IEVYNLMCDL 1.624 526LEQVNQMLNL 1.624 167 NMYDVNLNKN 1.624 484 KVPFYEPSHA 1.521 314GMDQTYCNKM 1.435 123 NINKLKTCGI 1.435 317 QTYCNKMEYM 1.369 128KTCGIHSKYM 1.328 508 LPTESLDCFC 1.243 358 FNSEEIVRNL 1.210 284VVDHAFGMLM 1.123 111 AEYLYTWDTL 1.107 805 KTYLPTFETT 1.079 466RIQPAPNNGT 1.025 139 AMYPTKTFPN 0.999 329 YFPRINFFYM 0.962 640RTSDSQYDAL 0.894 170 DVNLNKNFSL 0.813 89 QSQCPEGFDL 0.809 28 TCVESTRIWM0.731 716 IPTHYFVVLT 0.723 306 NIILLADHGM 0.683 242 LLKWLDLPKA 0.680 662RKMWDYFHSV 0.679 504 FANPLPTESL 0.669 452 FENIEVYNLM 0.667 232SVPFEERIST 0.652 425 YNNEFRSMEA 0.612 564 LLYHREYVSG 0.608 511ESLDCFCPHL 0.603 198 TAMYQGLKAA 0.587 401 LFVDQQWLAV 0.572 399VHLFVDQQWL 0.513 63 KDCCADYKSV 0.507 665 WDYFHSVLLI 0.491 392 KNVRIDKVHL0.488 29 CVESTRIWMC 0.480 216 VAINGSFPSI 0.468 440 GPSFKEKTEV 0.454 773TAHIARVRDV 0.444 339 YEGPAPRIRA 0.444 646 YDALITSNLV 0.444 610RVPPSESQKC 0.435 V2-A0201-10mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 5; each start position is specified, the length of peptideis 10 amino acids, and the end position for each peptide is thestart position plus nine. 7 CLQRKDCCAD 0.015 6 DCLQRKDCCA 0.009 4SDDCLQRKDC 0.003 8 LQRKDCCADY 0.001 2 SCSDDCLQRK 0.001 5 DDCLQRKDCC0.000 1 CSCSDDCLQR 0.000 10 RKDCCADYKS 0.000 3 CSDDCLQRKD 0.000 9QRKDCCADYK 0.000 V3-HLA-A2-10mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 7; each start position is specified, the length of peptideis 10 amino acids, and the end position for each peptide is thestart position plus nine. 7 SCPGGKPEAL 0.068 9 PGGKPEALWV 0.055 6ESCPGGKPEA 0.002 5 VESCPGGKPE 0 8 CPGGKPEALW 0 1 RPTNVESCPG 0 3TNVESCPGGK 0 10 GGKPEALWVE 0 2 PTNVESCPGG 0 4 NVESCPGGKP 0V4-HLA-A2-10mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 9; each start position is specified, the length of peptideis 10 amino acids, and the end position for each peptide is thestart position plus nine. 2 TYLPTFETPI 0.02 1 KTYLPTFETP 0.002

TABLe XII Start Subsequence Score V1-HLA-A3-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 3; each start positionis specified, the length of peptide is 9 amino acids, and the endposition for each peptide is the start position plus eight. 167NMYDVNLNK 300.000 314 GMDQTYCNK 60.000 632 FLYPPASNR 45.000 242LLKWLDLPK 40.000 337 YMYEFPAPR 30.000 663 KMWDYFHSV 27.000 436 FLAHGPSFK20.000 136 YMRAMYPTK 20.000 105 SMDGFRAEY 18.000 120 LMPNINKLK 15.000155 GLYPESHGI 13.500 379 YLTPDLPKR 9.000 803 QLKTYLPTF 9.000 743VLPFIIPHR 9.000 291 MLMEFLKQR 6.750 245 WLDLPKAER 6.000 322 KMEYMTDYF6.000 102 ILFSMDGFR 6.000 672 LLIKHATER 6.000 325 YMTDYFPRI 5.400 653NLVPMYEEF 4.500 32 STRIWMCNK 4.500 685 VVSGPIFDY 4.050 387 RLHYAKNVR4.000 610 RVPPSESQK 3.000 400 HLFVDQQWL 3.000 281 ALQVVDHAF 3.000 113YLYTWDTLM 3.000 459 NLMCDLLRI 2.700 618 KCSFYLADK 2.700 783 ELLTGLDFY2.700 119 TLMPNINKL 2.025 144 KTFPNHYTI 2.025 317 QTYCNKMEY 2.000 754NVESCPEGK 2.000 363 IVRNLSCRK 2.000 350 NIPHDFFSF 1.800 431 SMEAIFLAH1.800 203 GLKAATYFW 1.800 512 SLDCFCPHL 1.800 300 NLHNCVNII 1.800 807YLPTFETTI 1.800 740 WLDVLPFII 1.800 787 GLDFYQDKV 1.800 722 VVLTSCKNK1.500 128 KTCGIHSKY 1.350 118 DTLMPNINK 1.350 654 LVPMYEEFR 1.200 34RIWMCNKFR 1.000 272 GPVSARVIK 0.900 655 VPMYEEFRK 0.900 405 QQWLAVRSK0.900 197 LTAMYQGLK 0.900 568 REYVSGFGK 0.900 525 QLEQVNQML 0.900 199AMYQGLKAA 0.750 797 PVSEILQLK 0.675 532 MLNLTQEEI 0.600 5 GLENCRCDV0.600 8 NCRCDVACK 0.600 564 LLYHREYVS 0.600 555 VLQKNVDHC 0.600 384LPKRLHYAK 0.600 708 HLANTDVPI 0.600 650 ITSNLVPMY 0.600 196 WLTAMYQGL0.600 390 YAKNVRIDK 0.600 542 ATVKVNLPF 0.450 101 VILFSMDGF 0.450 794KVQPVSEIL 0.405 534 NLTQEEITA 0.400 54 SCSDDCLQK 0.400 622 YLADKNITH0.400 805 KTYLPTFET 0.338 79 WLEENCDTA 0.300 579 RMPMWSSYT 0.300 47RLEASLCSC 0.300 563 CLLYHREYV 0.300 577 AMRMPMWSS 0.270 362 EIVRNLSCR0.270 217 AINGSFPSI 0.270 482 LLKVPFYEP 0.270 500 SVCGFANPL 0.270 269HAGGPVSAR 0.270 126 KLKTCGIHS 0.240 474 GTHGSLNHL 0.203 51 SLCSCSDDC0.200 258 TMYFEEPDS 0.200 547 NLPFGRPRV 0.200 59 CLQKKDCCA 0.200 550FGRPRVLQK 0.180 290 GMLMEGLKQ 0.180 484 KVPFYEPSH 0.180 371 KPDQHFKPY0.180 55 CSDDCLQKK 0.150 139 AMYPTKTFP 0.150 450 EPEENIEVY 0.135 481HLLKVPFYE 0.135 241 TLLKWLDLP 0.135 456 EVYNLMCDL 0.135V2-HLA-A3-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 5; each start position is specified, the length of peptideis 9 amino acids, and the end position for each peptide is thestart position plus eight. 6 CLQRKDCCA 0.200 2 CSDDCLQRK 0.150 1SCSDDCLQR 0.080 9 RKDCCADYK 0.020 8 QRKDCCADY 0.004 5 DCLQRKDCC 0.001 7LQRKDCCAD 0.001 4 DDCLQRKDC 0.000 3 SDDCLQRKD 0.000V3-HLA-A3-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 7; each start position is specified, the length of peptideis 9 amino acids, and the end position for each peptide is thestart position plus eight. 3 NVESCPGGK 0.6 7 CPGGKPEAL 0.009 6 SCPGGKPEA0.003 9 GGKPEALWV 0.002 1 PTNVESCPG 0 2 TNVESCPGG 0 8 PGGKPEALW 0 4VESCPGGKP 0 5 ESCPGGKPE 0 V4-HLA-A3-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 9; each start positionis specified, the length of peptide is 9 amino acids, and the endposition for each peptide is the start position plus eight. 2 YLPTFETPI1.8 1 TYLPTFETP 0

TABLE XIII Start Subsequence Score V1-HLA-A3-10mers-161P2F10BEach peptide is a portion of SEQ ID NO: 3; each start positionis specified, the length of peptide is 10 amino acids, and the endposition for each peptide is the start position plus nine. 126KLKTCGIHSK 90.000 241 TLLKWLDLPK 60.000 119 TLMPNINKLK 33.750 663KMWDYFHSVL 27.000 653 NLVPMYEEFR 27.000 383 DLPKRLHYAK 18.000 196WLTAMYQGLK 18.000 290 GMLMEGLKQR 13.500 377 KPYLTPDLPK 9.000 337YMYEGPAPRI 6.750 654 LVPMYEEFRK 6.000 671 VLLIKHATER 6.000 155GLYPESHGII 4.050 684 NVVSGPIFDY 4.050 577 AMRMPMWSSY 4.000 102ILFSMDGFRA 3.000 785 LTGLDFYQDK 3.000 765 ALWVEERFTA 3.000 400HLFVDQQWLA 3.000 478 SLNHLLKVPF 2.000 226 YMPYNGSVPF 2.000 559NVDHCLLYHR 1.800 314 GMDQTYCNKM 1.800 300 NLHNCVNIIL 1.800 402FVDQQWLAVR 1.800 217 AINGSFPSIY 1.800 721 FVVLTSCKNK 1.500 220GSFPSIYMPY 1.350 543 TVKVNLPFGR 1.200 649 LITSNLVPMY 1.200 330FPRINFEYMY 1.080 762 KPEALWVEER 1.080 434 AIFLAHGPSF 1.000 531QMLNLTQEEI 0.900 105 SMDGFRAEYL 0.900 295 GLKQRNLHNC 0.900 555VLQKNVDHCL 0.900 362 EIVRNLSCRK 0.900 536 TQEEITATVK 0.900 632FLYPPASNRT 0.750 796 QPVSEILQLK 0.675 97 DLPPVILFSM 0.608 742 DVLPFIIPHR0.608 51 SLCSCSDDCL 0.600 682 GVNVVSGPIF 0.600 579 RMPMWSSYTV 0.600 5GLENCRCDVA 0.600 387 RLHYAKNVRI 0.600 247 DLPKAERPRF 0.600 570YVSGFGKAMR 0.600 393 NVRIDKVHLF 0.600 199 AMYQGLKAAT 0.500 139AMYPTKTFPN 0.450 648 ALITSNLVPM 0.450 379 YLTPDLPKRL 0.450 437LAHGPSFKEK 0.450 802 LQLKTYLPTF 0.405 162 GIIDNNMYDV 0.405 481HLLKVPFYEP 0.405 446 KTEVEPFENI 0.405 545 KVNLPFGRPR 0.360 192GQPMWLTAMY 0.360 34 RIWMCNKFRC 0.300 326 MTDYFPRINF 0.300 711 NTDVPIPTHY0.300 54 SCSDDCLQKK 0.300 507 PLPTESLDCF 0.300 136 YMRAMYPTKT 0.300 242LLKWLDLPKA 0.300 686 VSGPIFDYNY 0.270 784 LLTGLDFYQD 0.270 767WVEERFTAHI 0.270 172 NLNKNFSLSS 0.240 656 PMYEEFRKMW 0.225 115YTWDTLMPNI 0.225 805 KTYLPTFETT 0.225 144 KTFPNHYTIV 0.225 366NLSCRKPDQH 0.200 801 ILQLKTYLPT 0.200 356 FSFNSEEIVR 0.200 53 CSCSDDCLQK0.200 120 LMPNINKLKT 0.200 258 TMYFEEPDSS 0.200 368 SCRKPDQHFK 0.200 113YLYTWDTLMP 0.200 166 NNMYDVNLNK 0.180 495 EVSKFSVCGF 0.180 740WLDVLPFIIP 0.180 323 MEYMTDYFPR 0.180 563 CLLYHREYVS 0.180 101VILFSMDGFR 0.180 203 GLKAATYFWP 0.180 322 KMEYMTDYFP 0.180 534NLTQEEITAT 0.150 167 NMYDVNLNKN 0.150 309 LLADHGMDQT 0.150 596PLPPTVPDCL 0.135 280 KALQVVDHAF 0.135 31 ESTRIWMCNK 0.135 474 GTHGSLNHLL0.135 V2-HLA-A3-10mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 5; each start position is specified, the length of peptideis 10 amino acids, and the end position for each peptide is thestart position plus nine. 2 SCSDDCLQRK 0.300 8 LQRKDCCADY 0.120 1CSCSDDCLQR 0.040 7 CLQRKDCCAD 0.020 9 QRKDCCADYK 0.020 6 DCLQRKDCCA0.001 10 RKDCCADYKS 0.000 4 SDDCLQRKDC 0.000 5 DDCLQRKDCC 0.000 3CSDDCLQRKD 0.000 V3-HLA-A3-10mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 7; each start position is specified, the length of peptideis 10 amino acids, and the end position for each peptide is thestart position plus nine. 3 TNVESCPGGK 0.027 7 SCPGGKPEAL 0.009 8CPGGKPEALW 0.005 4 NVESCPGGKP 0.001 6 ESCPGGKPEA 0 10 GGKPEALWVE 0 1RPTNVESCPG 0 2 PTNVESCPGG 0 9 PGGKPEALWV 0 5 VESCPGGKPE 0V4-HLA-A3-10mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 9; each start position is specified, the length of peptideis 10 amino acids, and the end position for each peptide is thestart position plus nine. 1 KTYLPTFETP 0.045 2 TYLPTFETPI 0.004

TABLE XIV Start Subsequence Score V1-HLA-A11-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 3; each start positionis specified, the length of peptide is 9 amino acids, and the endposition for each peptide is the start position plus eight. 610RVPPSESQK 6.000 363 IVRNLSCRK 2.000 754 NVESCPEGK 2.000 167 NMYDVNLNK1.600 722 VVLTSCKNK 1.500 314 GMDQTYCNK 1.200 655 VPMYEEFRK 1.200 568REYVSGFGK 1.080 32 STRIWMCNK 1.000 197 LTAMYQGLK 1.000 272 GPVSARVIK0.900 118 DTLMPNINK 0.900 242 LLKWLDLPK 0.800 618 KCSFYLADK 0.600 390YAKNVRIDK 0.400 654 LVPMYEEFR 0.400 136 YMRAMYPTK 0.400 384 LPKRLHYAK0.400 436 FLAHGPSFK 0.400 54 SCSDDCLQK 0.400 667 YFHSVLLIK 0.400 720YFVVLTSCK 0.300 387 RLHYAKNVR 0.240 34 RIWMCNKFR 0.240 797 PVSEILQLK0.200 8 NCRCDVACK 0.200 120 LMPNINKLK 0.200 632 FLYPPASNR 0.160 337YMYEGPAPR 0.160 102 ILFSMDGFR 0.160 324 EYMTDYFPR 0.144 405 QQWLAVRSK0.120 378 PYLTPDLPK 0.120 144 KTFPNHYTI 0.120 672 LLIKHATER 0.120 554RVLQKNVDH 0.090 283 QVVDHAFGM 0.090 277 RVIKALQVV 0.090 743 VLPFIIPHR0.080 291 MLMEGLKQR 0.080 357 SFNSEEIVR 0.080 379 YLTPDLPKR 0.080 245WLDLPKAER 0.080 62 KKDCCADYK 0.060 794 KVQPVSEIL 0.060 537 QEEITATVK0.060 682 GVNVVSGPI 0.060 484 KVPFYEPSH 0.060 10 RCDVACKDR 0.060 685VVSGPIFDY 0.060 640 RTSDSQYDA 0.060 2 SFRGLENCR 0.040 476 HGSLNHLLK0.040 289 FGMLMEGLK 0.040 559 NVDHCLLYH 0.040 317 QTYCNKMEY 0.040 699FDAPDEITK 0.040 29 CVESTRIWM 0.040 550 FGRPRVLQK 0.040 402 FVDQQWLAV0.040 269 HAGGPVSAR 0.040 236 EERISTLLK 0.036 362 EIVRNLSCR 0.036 786TGLDFYQDK 0.030 240 STLLKWLDL 0.030 128 KTCGIHSKY 0.030 474 GTHGSLNHL0.030 542 ATVKVNLPF 0.030 458 YNLMCDLLR 0.024 663 KMWDYFHSV 0.024 131GIHSKYMRA 0.024 345 RIRAHNIPH 0.024 155 GLYPESHGI 0.024 203 GLKAATYFW0.024 395 RIDKVHLFV 0.024 393 NVRIDKVHL 0.020 284 VVDHAFGML 0.020 500SVCGFANPL 0.020 772 FTAHIARVR 0.020 369 CRKPDQHFK 0.020 71 SVCQGETSW0.020 55 CSDDCLQKK 0.020 127 LKTCGIHSK 0.020 767 WVEERFTAH 0.020 174NKNFSLSSK 0.020 544 VKVNLPFGR 0.018 742 DVLPFIIPH 0.018 805 KTYLPTFET0.018 192 GQPMWLTAM 0.018 90 SQCPEGFDL 0.018 297 KQRNLHNCV 0.018 459NLMCDLLRI 0.016 130 CGIHSKYMR 0.012 5 GLENCRCDV 0.012 42 RCGETRLEA 0.012253 RPRFYTMYF 0.012 740 WLDVLPFII 0.012 787 GLDFYQDKV 0.012 616SQKCSFYLA 0.012 350 NIPHDFFSF 0.012 V2-HLA-A11-9-mers-161P2F1OBEach peptide is a portion of SEQ ID NO: 5; each start positionis specified, the length of peptide is 9 amino acids, and the endposition for each peptide is the start position plus eight. 1 SCSDDCLQR0.080 9 RKDCCADYK 0.060 2 CSDDCLQRK 0.020 6 CLQRKDCCA 0.004 7 LQRKDCCAD0.001 8 QRKDCCADY 0.000 5 DCLQRKDCC 0.000 4 DDCLQRKDC 0.000 3 SDDCLQRKD0.000 V3-HLA-A11-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 7; each start position is specified, the length of peptideis 9 amino acids, and the end position for each peptide is thestart position plus eight. 3 NVESCPGGK 2 7 CPGGKPEAL 0.002 6 SCPGGKPEA0.002 9 GGKPEALWV 0.001 1 PTNVESCPG 0 2 TNVESCPGG 0 4 VESCPGGKP 0 8PGGKPEALW 0 5 ESCPGGKPE 0 V4-HLA-A11-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 9; each start positionis specified, the length of peptide is 9 amino acids, and the endposition for each peptide is the start position plus eight. 2 YLPTFETPI0.004 1 TYLPTFETP 0.001

TABLe XV Start Subsequence Score V1-HLA-A11-10mers-161P2F10BEach peptide is a portion of SEQ ID NO: 3; each start positionis specified, the length of peptide is 10 amino acids, and the endposition for each peptide is the start position plus nine. 654LVPMYEEFRK 6.000 377 KPYLTPDLPK 2.400 135 KYMRAMYPTK 2.400 721FVVLTSCKNK 1.500 543 TVKVNLPFGR 1.200 241 TLLKWLDLPK 1.200 126KLKTCGIHSK 1.200 785 LTGLDFYQDK 1.000 559 NVDHCLLYHR 0.800 719HYFVVLTSCK 0.800 389 HYAKNVRIDK 0.800 536 TQEEITATVK 0.600 666DYFHSVLLIK 0.480 196 WLTAMYQGLK 0.400 402 FVDQQWLAVR 0.400 570YVSGFGKAMR 0.400 119 TLMPNINKLK 0.400 698 HFDAPDEITK 0.400 435IFLAHGPSFK 0.300 796 QPVSEILQLK 0.300 383 DLPKRLHYAK 0.240 54 SCSDDCLQKK0.200 288 AFGMLMEGLK 0.200 368 SCRKPDQHFK 0.200 742 DVLPFIIPHR 0.180 631GFLYPPASNR 0.180 290 GMLMEGLKQR 0.180 362 EIVRNLSCRK 0.180 336FYMYEGPAPR 0.160 457 VYNLMCDLLR 0.160 166 NNMYDVNLNK 0.160 671VLLIKHATER 0.120 235 FEERISTLLK 0.120 545 KVNLPFGRPR 0.120 762KPEALWVEER 0.120 653 NLVPMYEEFR 0.120 101 VILFSMDGFR 0.120 437LAHGPSFKEK 0.100 684 NVVSGPIFDY 0.090 129 TCGIHSKYMR 0.080 323MEYMTDYFPR 0.072 567 HREYVSGFGK 0.060 753 TNVESCPEGK 0.060 271GGPVSARVIK 0.060 682 GVNVVSGPIF 0.060 489 EPSHAEEVSK 0.060 484KVPFYEPSHA 0.060 144 KTFPNHYTIV 0.060 398 KVHLFVDQQW 0.060 475THGSLNHLLK 0.040 117 WDTLMPNINK 0.040 173 LNKNFSLSSK 0.040 313HGMDQTYCNK 0.040 597 LPPTVPDCLR 0.040 284 VVDHAFGMLM 0.040  53CSCSDDCLQK 0.040 601 VPDCLRADVR 0.040 549 PEGRPRVLQK 0.040 162GIIDNNMYDV 0.036 609 VRVPPSESQK 0.030 283 QVVDHAFGML 0.030 640RTSDSQYDAL 0.030 446 KTEVEPFENI 0.030 474 GTHGSLNHLL 0.030 282LQVVDHAFGM 0.027 421 GNHGYNNEFR 0.024 663 KMWDYFHSVL 0.024 765ALWVEERFTA 0.024 579 RMPMWSSYTV 0.024 155 GLYPESHGII 0.024 102ILFSMDGFRA 0.024 152 IVTGLYPESH 0.020 71 SVCQGETSWL 0.020 304 CVNIILLADH0.020 207 ATYFWPGSEV 0.020 767 WVEERFTAHI 0.020 197 LTAMYQGLKA 0.020 600TVPDCLRADV 0.020 115 YTWDTLMPNI 0.020 617 QKCSFYLADK 0.020 326MTDYFPRINF 0.020 317 QTYCNKMEYM 0.020 61 QKKDCCADYK 0.020 393 NVRIDKVHLF0.020 386 KRLHYAKNVR 0.018 244 KWLDLPKAER 0.018 192 GQPMWLTAMY 0.018 272GPVSARVIKA 0.018 170 DVNLNKNFSL 0.018 404 DQQWLAVRSK 0.018 400HLFVDQQWLA 0.016 356 FSFNSEEIVR 0.016 128 KTCGIHSKYM 0.015 378PYLTPDLPKR 0.012 771 RFTAHIARVR 0.012 268 GHAGGPVSAR 0.012 250KAERPRFYTM 0.012 108 GFRAEYLYTW 0.012 573 GFGKAMRMPM 0.012 387RLHYAKNVRI 0.012 V2-HLA-A11-10mers-161P2F10BEach peptide is a portion of SEQ ID NO: 5; each start positionis specified, the length of peptide is 10 amino acids, and the endposition for each peptide is the start position plus nine. 2 SCSDDCLQRK0.200 9 QRKDCCADYK 0.020 1 CSCSDDCLQR 0.008 8 LQRKDCCADY 0.006 6DCLQRKDCCA 0.001 7 CLQRKDCCAD 0.000 10 RKDCCADYKS 0.000 4 SDDCLQRKDC0.000 5 DDCLQRKDCC 0.000 3 CSDDCLQRKD 0.000 V3-HLA-A11-10mers-161P2F10BEach peptide is a portion of SEQ ID NO: 7; each start positionis specified, the length of peptide is 10 amino acids, and the endposition for each peptide is the start position plus nine. 3 TNVESCPGGK0.06 8 CPGGKPEALW 0.002 7 SCPGGKPEAL 0.002 4 NVESCPGGKP 0.002 1RPTNVESCPG 0.001 10 GGKPEALWVE 0 2 PTNVESCPGG 0 6 ESCPGGKPEA 0 9PGGKPEALWV 0 5 VESCPGGKPE 0 V4-HLA-A11-10mers-161P2F10BEach peptide is a portion of SEQ ID NO: 9; each start positionis specified, the length of peptide is 10 amino acids, and the endposition for each peptide is the start position plus nine. 2 TYLPTFETPI0.006 1 KTYLPTFETP 0.006

TABLE XVI Start Subsequence Score V1-HLA-A24-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 3; each start positionis specified, the length of peptide is 9 amino acids, and the endposition for each peptide is the start position plus eight. 112EYLYTWDTL 300.000 457 VYNLMCDLL 300.000 328 DYFPRINFF 144.000 156LYPESHGII 90.000 338 MYEGPAPRI 75.000 666 DYFHSVLLI 50.000 424 GYNNEFRSM45.000 40 KFRCGETRL 40.000 318 TYCNKMEYM 25.000 288 AFGMLMEGL 24.000 794KVQPVSEIL 20.160 95 GFDLPPVIL 20.000 435 IFLAHGPSF 15.000 135 KYMRAMYPT15.000 633 LYPPASNRT 10.800 790 FYQDKVQPV 10.800 806 TYLPTFETT 10.800359 NSEEIVRNL 10.080 525 QLEQVNQML 10.080 660 EFRKMWDYF 10.000 428EFRSMEAIF 10.000 569 EYVSGFGKA 9.900 238 RISTLLKWL 9.600 119 TLMPNINKL9.504 657 MYEEFRKMW 9.000 621 FYLADKNIT 9.000 225 IYMPYNGSV 9.000 200MYQGLKAAT 9.000 380 LTPDLPKRL 8.640 597 LPPTVPDCL 8.400 140 MYPTKTFPN7.500 800 EILQLKTYL 7.200 149 HYTIVTGLY 7.000 719 HYFVVLTSC 7.000 701APDEITKHL 6.720 168 MYDVNLNKN 6.600 736 NCPGWLDVL 6.000 259 MYFEEPDSS6.000 138 RAMYPTKTF 6.000 758 CPEGKPEAL 6.000 733 TPENCPGWL 6.000 527EQVNQMLNL 6.000 240 STLLKWLDL 6.000 615 ESQKCSFYL 6.000 165 DNNMYDVNL6.000 72 VCQGETSWL 6.000 322 KMEYMTDYF 6.000 645 QYDALITSN 6.000 796QPVSEILQL 6.000 171 VNLNKNFSL 6.000 505 ANPLPTESL 6.000 556 LQKNVDHCL5.600 347 RAHNIPHDF 5.600 540 ITATVKVNL 5.600 274 VSARVIKAL 5.600 208TYFWPGSEV 5.500 355 FESENSEEI 5.500 620 SFYLADKNI 5.000 474 GTHGSLNHL4.800 456 EVYNLMCDL 4.800 716 IPTHYFVVL 4.800 196 WLTAMYQGL 4.800 90SQCPEGFDL 4.800 500 SVCGFANPL 4.800 400 HLFVDQQWL 4.800 284 VVDHAFGML4.800 776 IARVRDVEL 4.400 542 ATVKVNLPF 4.200 764 EALWVEERF 4.200 281ALQVVDHAF 4.200 189 WWHGQPMWL 4.000 512 SLDCFCPHL 4.000 393 NVRIDKVHL4.000 583 WSSYTVPQL 4.000 302 HNCVNIILL 4.000 664 MWDYFHSVL 4.000 548LPFGRPRVL 4.000 52 LCSCSDDCL 4.000 253 RPRFYTMYF 4.000 641 TSDSQYDAL4.000 653 NLVPMYEEF 3.960 234 PFEERISTL 3.600 350 NIPHDFFSF 3.600 101VILFSMDGF 3.000 299 RNLHNCVNI 3.000 713 DVPIPTHYF 3.000 683 VNVVSGPIF3.000 202 QGLKAATYF 3.000 421 GNHGYNNEF 2.640 739 GWLDVLPFI 2.520 144KTFPNHYTI 2.400 368 SCRKPDQHF 2.400 479 LNHLLKVPF 2.400 508 LPTESLDCF2.400 682 GVNVVSGPI 2.100 88 QQSQCPEGF 2.000 227 MPYNGSVPF 2.000 496VSKFSVCGF 2.000 248 LPKAERPRF 2.000 803 QLKTYLPTF 2.000V2-HLA-A24-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 5; each start position is specified, the length of peptideis 9 amino acids, and the end position for each peptide is thestart position plus eight. 5 DCLQRKDCC 0.150 6 CLQRKDCCA 0.150 2CSDDCLQRK 0.014 8 QRKDCCADY 0.012 1 SCSDDCLQR 0.012 4 DDCLQRKDC 0.010 7LQRKDCCAD 0.010 9 RKDCCADYK 20 0.002 3 SDDCLQRKD 0.001V3-HLA-A24-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 7; each start position is specified, the length of peptideis 9 amino acids, and the end position for each peptide is thestart position plus eight. 7 CPGGKPEAL 4 6 SCPGGKPEA 0.165 9 GGKPEALWV0.12 2 TNVESCPGG 0.018 3 NVESCPGGK 0.015 5 ESCPGGKPE 0.012 8 PGGKPEALW0.01 1 PTNVESCPG 0.02 4 VESCPGGKP 0.001 V4-HLA-A24-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 9; each start positionis specified, the length of peptide is 9 amino acids, and the endposition for each peptide is the start position plus eight. 2 YLPTFETPI1.5 1 TYLPTFETP 1.08

TABLE XVII Start Subsequence Score V1-HLA-A24-10mers-161P2F10BEach peptide is a portion of SEQ ID NO: 3; each start positionis specified, the length of peptide is 10 amino acids, and the endposition for each peptide is the start position plus nine. 645QYDALITSNL 280.000 168 MYDVNLNKNF 120.000 565 LYHREYVSGF 100.000 806TYLPTFETTI 90.000 324 EYMTDYFPRI 90.000 569 EYVSGFGKAM 37.500 112EYLYTWDTLM 37.500 375 HFKPYLTPDL 28.800 428 EFRSMEAIFL 20.000 790FYQDKVQPVS 12.600 524 TQLEQVNQML 12.096 392 KNVRIDKVHL 12.000 700DAPDEITKHL 10.080 690 IFDYNYDGHF 10.000 95 GFDLPPVILF 10.000 487FYEPSHAEEV 9.900 663 KMWDYFHSVL 9.600 640 RTSDSQYDAL 9.600 633LYPPASNRTS 9.000 283 QVVDHAFGML 8.640 328 DYFPRINFFY 8.400 280KALQVVDHAF 8.400 555 VLQKNVDHCL 8.400 371 KPDQHFKPYL 8.000 118DTLMPNINKL 7.920 200 MYQGLKAATY 7.500 692 DYNYDGHFDA 7.500 757SCPEGKPEAL 7.200 504 FANPLPTESL 7.200 511 ESLDCFCPHL 7.200 195MWLTAMYQGL 7.200 732 HTPENCPGWL 7.200 499 FSVCGFANPL 7.200 43 CGETRLEASL7.200 358 FNSEEIVRNL 6.720 547 NLPFGRPRVL 6.000 170 DVNLNKNFSL 6.000 795VQPVSEILQL 6.000 89 QSQCPEGFDL 6.000 470 APNNGTHGSL 6.000 209 YFWPGSEVAI6.000 588 VPQLGDTSPL 6.000 292 LMEGLKQRNL 6.000 379 YLTPDLPKRL 5.760 300NLHNCVNIIL 5.600 539 EITATVKVNL 5.600 68 DYKSVCQGET 5.500 354 DFFSFNSEEI5.500 234 PFEERISTLL 5.040 114 LYTWDTLMPN 5.000 318 TYCNKMEYMT 5.000 208TYFWPGSEVA 5.000 585 SYTVPQLGDT 5.000 735 ENCPGWLDVL 4.800 287HAFGMLMEGL 4.800 473 NGTHGSLNHL 4.800 474 GTHGSLNHLL 4.800 233VPFEERISTL 4.800 94 EGFDLPPVIL 4.800 329 YFPRINFFYM 4.500 775 HIARVRDVEL4.400 349 HNIPHDFFSF 4.320 213 GSEVAINGSF 4.200 51 SLCSCSDDCL 4.000 71SVCQGETSWL 4.000 239 ISTLLKWLDL 4.000 517 CPHLQNSTQL 4.000 582MWSSYTVPQL 4.000 188 AWWHGQPMWL 4.000 776 IARVRDVELL 4.000 347RAHNIPHDFF 4.000 105 SMDGFRAEYL 4.000 556 LQKNVDHCLL 4.000 456EVYNLMCDLL 4.000 664 MWDYFHSVLL 4.000 420 GGNHGYNNEF 3.960 478SLNHLLKVPF 3.600 299 RNLHNCVNII 3.600 446 KTEVEPFENI 3.600 652SNLVPMYEEF 3.300 247 DLPKAERPRF 3.000 802 LQLKTYLPTF 3.000 87 AQQSQCPEGF3.000 226 YMPYNGSVPF 3.000 451 PFENIEVYNL 3.000 682 GVNVVSGPIF 3.000 781DVELLTGLDF 3.000 623 LADKNITHGF 2.800 541 TATVKVNLPF 2.800 32 STRIWMCNKF2.640 573 GFGKAMRMPM 2.500 367 LSCRKPDQHF 2.400 739 GWLDVLPFII 2.160 681NGVNVVSGPI 2.100 434 AIFLAHGPSF 2.000 393 NVRIDKVHLF 2.000 737CPGWLDVLPF 2.000 495 EVSKFSVCGF 2.000 326 MTDYFPRINF 2.000 201YQGLKAATYF 2.000 V2-HLA-A24-10mers-161P2F10BEach peptide is a portion of SEQ ID NO: 5; each start positionis specified, the length of peptide is 10 amino acids, and the endposition for each peptide is the start position plus nine. 6 DCLQRKDCCA0.150 8 LQRKDCCADY 0.100 10 RKDCCADYKS 0.022 3 CSDDCLQRKD 0.016 7CLQRKDCCAD 0.015 2 SCSDDCLQRK 0.014 4 SDDCLQRKDC 0.010 1 CSCSDDCLQR0.010 5 DDCLQRKDCC 0.010 9 QRKDCCADYK 0.001 V3-HLA-A24-10mers-161P2F10BEach peptide is a portion of SEQ ID NO: 7; each start positionis specified, the length of peptide is 10 amino acids, and the endposition for each peptide is the start position plus nine. 7 SCPGGKPEAL6 6 ESCPGGKPEA 0.132 8 CPGGKPEALW 0.1 1 RPTNVESCPG 0.02 3 TNVESCPGGK0.018 4 NVESCPGGKP 0.017 10 GGKPEALWVE 0.012 9 PGGKPEALWV 0.01 2PTNVESCPGG 0.002 5 VESCPGGKPE 0.001 2 TYLPTFETPI 90 1 KTYLPTFETP 0.024

TABLE XVIII Start Subsequence Score V1-HLA-B7-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 3; each start positionis specified, the length of peptide is 9 amino acids, and the endposition for each peptide is the start position plus eight. 343APRIRAHNI 240.000 330 FPRINFFYM 200.000 393 NVRIDKVHL 200.000 776IARVRDVEL 120.000 548 LPFGRPRVL 80.000 716 IPTHYFVVL 80.000 796QPVSEILQL 80.000 597 LPPTVPDCL 80.000 701 APDEITKHL 72.000 552 RPRVLQKNV40.000 758 CPEGKPEAL 24.000 733 TPENCPGWL 24.000 500 SVCGFANPL 20.000456 EVYNLMCDL 20.000 98 LPPVILFSM 20.000 794 KVQPVSEIL 20.000 505ANPLPTESL 18.000 580 MPMWSSYTV 12.000 119 TLMPNINKL 12.000 284 VVDHAFGML6.000 283 QVVDHAFGM 5.000 570 YVSGFGKAM 5.000 778 RVRDVELLT 5.000 274VSARVIKAL 4.000 240 STLLKWLDL 4.000 736 NCPGWLDVL 4.000 90 SQCPEGFDL4.000 714 VPIPTHYFV 4.000 800 EILQLKTYL 4.000 196 WLTAMYQGL 4.000 540ITATVKVNL 4.000 238 TISTLLKWL 4.000 253 RPRFYTMYF 4.000 72 VCQGETSWL4.000 474 GTHGSLNHL 4.000 40 KFRCGETRL 4.000 400 HLFVDQQWL 4.000 615ESQKCSFYL 4.000 171 VNLNKNFSL 4.000 527 EQVNQMLNL 4.000 165 DNNMYDVNL4.000 583 WSSYTVPQL 4.000 380 LTPDLPKRL 4.000 556 LQKNVDHCL 4.000 52LCSCSDDCL 4.000 302 HNCVNIILL 4.000 251 AERPRFYTM 3.000 233 VPFEERIST3.000 409 AVRSKSNTN 3.000 29 CVESTRIWM 2.250 146 FPNHYTIVT 2.000 682GVNVVSGPI 2.000 485 VPFYEPSHA 2.000 611 VPPSESQKC 2.000 297 KQRNLHNCV2.000 121 MPNINKLKT 2.000 601 VPDCLRADV 1.800 608 DVRVPPSES 1.500 574FGKAMRMPM 1.500 219 NGSFPSIYM 1.500 288 AFGMLMEGL 1.200 459 NLMCDLLRI1.200 525 QLEQVNQML 1.200 512 SLDCFCPHL 1.200 193 QPMWLTAMY 1.200 217AINGSFPSI 1.200 641 TSDSQYDAL 1.200 777 ARVRDVELL 1.200 359 NSEEIVRNL1.200 470 APNNGTHGS 1.200 453 ENIEVYNLM 1.000 307 IILLADHGM 1.000 113YLYTWDTLM 1.000 572 SGFGKAMRM 1.000 524 TQLEQVNQM 1.000 129 TCGIHSKYM1.000 192 GQPMWLTAM 1.000 649 LITSNLVPM 1.000 45 ETRLEASLC 1.000 277RVIKALQVV 1.000 198 TAMYQGLKA 0.900 577 AMRMPMWSS 0.900 248 LPKAERPRF0.600 655 VPMYEEFRK 0.600 647 DALITSNLV 0.600 270 AGGPVSARV 0.600 528QVNQMLNLT 0.500 363 IVRNLSCRK 0.500 670 SVLLIKHAT 0.500 13 VACKDRGDC0.450 589 PQLGDTSPL 0.400 708 HLANTDVPI 0.400 299 RNLHNCVNI 0.400 807YLPTFETTI 0.400 475 THGSLNHLL 0.400 452 FENIEVYNL 0.400 94 EGFDLPPVI0.400 227 MPYNGSVPF 0.400 471 PNNGTHGSL 0.400 211 WPGSEVAIN 0.400V2-HLA-B7-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 5; each start position is specified, the length of peptideis 9 amino acids, and the end position for each peptide is thestart position plus eight. 5 DCLQRKDCC 0.100 6 CLQRKDCCA 0.100 7LQRKDCCAD 0.100 4 DDCLQRKDC 0.015 1 SCSDDCLQR 0.010 2 CSDDCLQRK 0.003 8QRKDCCADY 0.002 3 SDDCLQRKD 0.000 9 RKDCCADYK 0.000V3-HLA-B7-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 7; each start position is specified, the length of peptideis 9 amino acids, and the end position for each peptide is thestart position plus eight. 7 CPGGKPEAL 80 9 GGKPEALWV 0.2 6 SCPGGKPEA0.1 3 NVESCPGGK 0.015 5 ESCPGGKPE 0.01 2 TNVESCPGG 0.01 8 PGGKPEALW0.003 4 VESCPGGKP 0.002 1 PTNVESCPG 0.001 V4-HLA-B7-9mers-161P2F1B8Each peptide is a portion of SEQ ID NO: 9; each start positionis specified, the length of peptide is 9 amino acids, and the endposition for each peptide is the start position plus eight. 2 YLPTFETPI0.4 1 TYLPTFETP 0.001

TABLE XIX Start Subsequence Score V1-HLA-B7-10mers-161P2F10BEach peptide is a portion of SEQ ID NO: 3; each start positionis specified, the length of peptide is 10 amino acids, and the endposition for each peptide is the start position plus nine. 470APNNGTHGSL 240.000 776 IARVRDVELL 120.000 588 VPQLGDTSPL 80.000 517CPHLQNSTQL 80.000 233 VPFEERISTL 80.000 655 VPMYEEFRKM 60.000 371KPDQHFKPYL 24.000 283 QVVDHAFGML 20.000 186 NPAWWHGQPM 20.000 170DVNLNKNFSL 20.000 456 EVYNLMCDLL 20.000 71 SVCQGETSWL 20.000 504FANPLPTESL 18.000 409 AVRSKSNTNC 15.000 287 HAFGMLMEGL 12.000 700DAPDEITKHL 12.000 94 EGFDLPPVIL 6.000 343 APRIRAHNIP 6.000 275SARVIKALQV 6.000 300 NLHNCVNIIL 4.000 89 QSQCPEGFDL 4.000 775 HIARVRDVEL4.000 239 ISTLLKWLDL 4.000 392 KNVRIDKVHL 4.000 440 GPSFKEKTEV 4.000 547NLPFGRPRVL 4.000 795 VQPVSEILQL 4.000 732 HTPENCPGWL 4.000 473NGTHGSLNHL 4.000 555 VLQKNVDHCL 4.000 511 ESLDCFCPHL 4.000 51 SLCSCSDDCL4.000 714 VPIPTHYFVV 4.000 757 SCPEGKPEAL 4.000 330 FRINFFYMY 4.000 663KMWDYFHSVL 4.000 640 RTSDSQYDAL 4.000 524 TQLEQVNQML 4.000 428EFRSMEAIFL 4.000 358 FNSEEIVRNL 4.000 735 ENCPGWLDVL 4.000 118DTLMPNINKL 4.000 539 EITATVKVNL 4.000 556 LQKNVDHCLL 4.000 474GTHGSLNHLL 4.000 499 FSVCGFANPL 4.000 379 YLTPDLPKRL 4.000 744LPFIIPHRPT 3.000 648 ALITSNLVPM 3.000 716 IPTHYFVVLT 2.000 552RPRVLQKNVD 2.000 595 SPLPPTVPDC 2.000 272 GPVSARVIKA 2.000 273PVSARVIKAL 2.000 508 LPTESLDCFC 2.000 253 RPRFYTMYFE 2.000 121MPNINKLKTC 2.000 506 NPLPTESLDC 2.000 701 APDEITKHLA 1.800 600TVPDCLRADV 1.500 218 INGSFPSIYM 1.500 28 TCVESTRIWM 1.500 284 VVDHAFGMLM1.500 43 CGETRLEASL 1.200 270 AGGPVSARVI 1.200 292 LMEGLKQRNL 1.200 105SMDGFRAEYL 1.200 624 ADKNITHGFL 1.200 188 AWWHGQPMWL 1.200 216VAINGSFPSI 1.200 92 CPEGFDLPPV 1.200 111 AEYLYTWDTL 1.200 571 VSGFGKAMRM1.000 423 HGYNNEFRSM 1.000 713 DVPIPTHYFV 1.000 191 HGQPMWLTAM 1.000 393NVRIDKVHLF 1.000 159 ESHGIIDNNM 1.000 523 STQLEQVNQM 1.000 282LQVVDHAFGM 1.000 97 DLPPVILFSM 1.000 136 YMRAMYPTKT 1.000 306 NIILLADHGM1.000 317 QTYCNKMEYM 1.000 131 GIHSKYMRAM 1.000 128 KTCGIHSKYM 1.000 207ATYFWPGSEV 0.900 198 TAMYQGLKAA 0.900 250 KAERPRFYTM 0.900 12 DVACKDRGDC0.750 232 SVPFEERIST 0.750 608 DVRVPPSESQ 0.750 337 YMYEGPAPRI 0.600 676HATERNGVNV 0.600 390 YAKNVRIDKV 0.600 773 TAHIARVRDV 0.600 193QPMWLTAMYQ 0.600 767 WVEERFTAHI 0.600 580 MPMWSSYTVP 0.600 269HAGGPVSARV 0.600 V2-HLA-B7-10mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 5; each start position is specified, the length of peptideis 10 amino acids, and the end position for each peptide is thestart position plus nine. 8 LQRKDCCADY 0.200 6 DCLQRKDCCA 0.100 7CLQRKDCCAD 0.010 1 CSCSDDCLQR 0.010 5 DDCLQRKDCC 0.010 2 SCSDDCLQRK0.010 4 SDDCLQRKDC 0.004 3 CSDDCLQRKD 0.003 9 QRKDCCADYK 0.001 10RKDCCADYKS 0.001 V3-HLA-B7-10mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 7; each start position is specified, the length of peptideis 10 amino acids, and the end position for each peptide is thestart position plus nine. 7 SCPGGKPEAL 4 8 CPGGKPEALW 0.6 1 RPTNVESCPG0.2 6 ESCPGGKPEA 0.1 4 NVESCPGGKP 0.023 9 PGGKPEALWV 0.02 10 GGKPEALWVE0.01 3 TNVESCPGGK 0.01 2 PTNVESCPGG 0.001 5 VESCPGGKPE 0.001V4-HLA-B7-10mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 9; each start position is specified, the length of peptideis 10 amino acids, and the end position for each peptide is thestart position plus nine. 2 TYLPTFETPI 0.04 1 KTYLPTFETP 0.01

TABLE XX Start Subsequence Score V1-HLA-B3501-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 3; each start positionis specified, the length of peptide is 9 amino acids, and the endposition for each peptide is the start position plus eight. 253RPRFYTMYF 120.000 330 FPRINFFYM 120.000 248 LPKAERPRF 90.000 450EPFENIEVY 80.000 98 LPPVILFSM 40.000 508 LPTESLDCF 40.000 193 QPMWLTAMY40.000 133 HSKYMRAMY 30.000 796 QPVSEILQL 30.000 552 RPRVLQKNV 24.000371 KPDQHFKPY 24.000 343 APRIRAHNI 24.000 597 LPPTVPDCL 20.000 548LPFGRPRVL 20.000 716 IPTHYFVVL 20.000 227 MPYNGSVPF 20.000 496 VSKFSVCGF15.000 701 APDEITKHL 12.000 776 IARVRDVEL 9.000 558 KNVDHCLLY 8.000 733TPENCPGWL 6.000 758 CPEGKPEAL 6.000 347 RAHNIPHDF 6.000 138 RAMYPTKTF6.000 638 SNRTSDSQY 6.000 233 VPFEERIST 6.000 574 FGKAMRMPM 6.000 583WSSYTVPQL 5.000 615 ESQKCSFYL 5.000 274 VSARVIKAL 5.000 393 NVRIDKVHL4.500 453 ENIEVYNLM 4.000 128 KTCGIHSKY 4.000 580 MPMWSSYTV 4.000 714VPIPTHYFV 4.000 351 IPHDFFSFN 4.000 626 KNITHGFLY 4.000 283 QVVDHAFGM4.000 524 TQLEQVNQM 4.000 803 QLKTYLPTF 3.000 611 VPPSESQKC 3.000 368SCRKPDQHF 3.000 359 NSEEIVRNL 3.000 556 LQKNVDHCL 3.000 764 EALWVEERF3.000 485 VPFYEPSHA 3.000 161 HGIIDNNMY 3.000 180 SSKEQNNPA 3.000 211WPGSEVAIN 3.000 238 RISTLLKWL 2.000 222 FPSIYMPYN 2.000 562 HCLLYHREY2.000 685 VVSGPIFDY 2.000 649 LITSNLVPM 2.000 650 ITSNLVPMY 2.000 146FPNHYTIVT 2.000 218 INGSFPSIY 2.000 634 YPPASNRTS 2.000 107 DGFRAEYLY2.000 783 ELLTGLDFY 2.000 570 YVSGFGKAM 2.000 121 MPNINKLKT 2.000 113YLYTWDTLM 2.000 317 QTYCNKMEY 2.000 231 GSVPFEERI 2.000 417 NCGGGNHGY2.000 219 NGSFPSIYM 2.000 129 TCGIHSKYM 2.000 201 YQGLKAATY 2.000 307IILLADHGM 2.000 687 SGPIFDYNY 2.000 192 GQPMWLTAM 2.000 489 EPSHAEEVS2.000 794 KVQPVSEIL 2.000 470 APNNGTHGS 2.000 572 SGFGKAMRM 2.000 430RSMEAIFLA 2.000 380 LTPDLPKRL 2.000 641 TSDSQYDAL 1.500 203 GLKAATYFW1.500 350 NIPHDFFSF 1.500 400 HLFVDQQWL 1.500 90 SQCPEGFDL 1.500 72VCQGETSWL 1.500 124 INKLKTCGI 1.200 61 QKKDCCADY 1.200 778 RVRDVELLT1.200 601 VPDCLRADV 1.200 297 KQRNLHNCV 1.200 736 NCPGWLDVL 1.000 732HTPENCPGW 1.000 196 WLTAMYQGL 1.000 28 TCVESTRIW 1.000 683 VNVVSGPIF1.000 202 QGLKAATYF 1.000 500 SVCGFANPL 1.000 527 EQVNQMLNL 1.000 88QQSQCPEGF 1.000 713 DVPIPTHYF 1.000 302 HNCVNIILL 1.000V2-HLA-B3501-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 5; each start position is specified, the length of peptideis 9 amino acids, and the end position for each peptide is thestart position plus eight. 8 QRKDCCADY 1.200 5 DCLQRKDCC 0.100 6CLQRKDCCA 0.100 7 LQRKDCCAD 0.045 1 SCSDDCLQR 0.030 2 CSDDCLQRK 0.030 4DDCLQRKDC 0.010 9 RKDCCADYK 0.001 3 SDDCLQRKD 0.000V3-HLA-B3501-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 7; each start position is specified, the length of peptideis 9 amino acids, and the end position for each peptide is thestart position plus eight. 7 CPGGKPEAL 20 9 GGKPEALWV 0.9 6 SCPGGKPEA0.1 8 PGGKPEALW 0.05 5 ESCPGGKPE 0.05 2 TNVESCPGG 0.02 3 NVESCPGGK 0.0031 PTNVESCPG 0.002 4 VESCPGGKP 0.001 V4-HLA-B3501-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 9; each start positionis specified, the length of peptide is 9 amino acids, and the endposition for each peptide is the start position plus eight. 2 YLPTFETPI0.4 1 TYLPTFETP 0.001

TABLE XXI V1-HLA-B35-10mers-161P2F10BEach peptide is a portion of SEQ ID NO: 3; eachstart position is specified, the length of peptideis 10 amino acids, and the end position for eachpeptide is the start position plus nine. Start Subsequence Score 248LPKAERPRFY 120.000 330 FPRINFFYMY 120.000 655 VPMYEEFRKM 60.000 233VPFEERISTL 40.000 141 YPTKTFPNHY 40.000 186 NPAWWHGQPM 40.000 737CPGWLDVLPF 30.000 588 VPQLGDTSPL 30.000 104 FSMDGFRAEY 20.000 470APNNGTHGSL 20.000 517 CPHLQNSTQL 20.000 180 SSKEQNNPAW 15.000 776IARVRDVELL 13.500 371 KPDQHFKPYL 12.000 381 TPDLPKRLHY 12.000 686VSGPIFDYNY 10.000 220 GSFPSIYMPY 10.000 571 VSGFGKAMRM 10.000 511ESLDCFCPHL 10.000 159 ESHGIIDNNM 10.000 637 ASNRTSDSQY 10.000  89QSQCPEGFDL 7.500 280 KALQVVDHAF 6.000 700 DAPDEITKHL 6.000 347RAHNIPHDFF 6.000 577 AMRMPMWSSY 6.000 320 CNKMEYMTDY 6.000 440GPSFKEKTEV 6.000 384 LPKRLHYAKN 6.000  60 LQKKDCCADY 6.000 499FSVCGFANPL 5.000 239 ISTLLKWLDL 5.000 367 LSCRKPDQHF 5.000  70KSVCQGETSW 5.000 556 LQKNVDHCLL 4.500 612 PPSESQKCSF 4.000 640RTSDSQYDAL 4.000 351 IPHDFFSFNS 4.000 508 LPTESLDCFC 4.000 663KMWDYFHSVL 4.000 714 VPIPTHYFVV 4.000  28 TCVESTRIWM 4.000 450EPFENIEVYN 4.000 128 KTCGIHSKYM 4.000 250 KAERPRFYTM 3.600 287HAFGMLMEGL 3.000  32 STRIWMCNKF 3.000 393 NVRIDKVHLF 3.000  14ACKDRGDCCW 3.000 392 KNVRIDKVHL 3.000 758 CPEGKPEALW 3.000 541TATVKVNLPF 3.000 423 HGYNNEFRSM 3.000 506 NPLPTESLDC 3.000 798VSEILQLKTY 3.000 504 FANPLPTESL 3.000  97 DLPPVILFSM 2.000 316DQTYCNKMEY 2.000 595 SPLPPTVPDC 2.000 272 GPVSARVIKA 2.000 648ALITSNLVPM 2.000 479 LNHLLKVPFY 2.000 283 QVVDHAFGML 2.000 416TNCGGGNHGY 2.000 131 GIHSKYMRAM 2.000 649 LITSNLVPMY 2.000 684NVVSGPIFDY 2.000 306 NIILLADHGM 2.000 121 MPNINKLKTC 2.000 358FNSEEIVRNL 2.000 218 INGSFPSIYM 2.000 192 GQPMWLTAMY 2.000 317QTYCNKMEYM 2.000 757 SCPEGKPEAL 2.000 611 VPPSESQKCS 2.000 744LPFIIPHRPT 2.000 217 AINGSFPSIY 2.000 191 HGQPMWLTAM 2.000 341GPAPRIRAHN 2.000 732 HTPENCPGWL 2.000 282 LQVVDHAFGM 2.000 716IPTHYFVVLT 2.000  94 EGFDLPPVIL 2.000 523 STQLEQVNQM 2.000 619CSFYLADKNI 2.000 524 TQLEQVNQML 2.000 748 IPHRPTNVES 2.000 390YAKNVRIDKV 1.800 310 LADHGMDQTY 1.800 275 SARVIKALQV 1.800  92CPEGFDLPPV 1.800  71 SVCQGETSWL 1.500 213 GSEVAINGSF 1.500 349HNIPHDFFSF 1.500 795 VQPVSEILQL 1.500 574 FGKAMRMPMW 1.500 496VSKFSVCGFA 1.500 247 DLPKAERPRF 1.500 552 RPRVLQKNVD 1.200 676HATERNGVNV 1.200 V2-HLA-B35-10mers-161P2F10BEach peptide is a portion of SEQ ID NO: 5; eachstart position is specified, the length of peptideis 10 amino acids, and the end position for eachpeptide is the start position plus nine. Start Subsequence Score 8LQRKDCCADY 6.000 6 DCLQRKDCCA 0.100 1 CSCSDDCLQR 0.075 3 CSDDCLQRKD0.030 2 SCSDDCLQRK 0.020 7 CLQRKDCCAD 0.015 5 DDCLQRKDCC 0.010 9QRKDCCADYK 0.006 10  RKDCCADYKS 0.006 4 SDDCLQRKDC 0.003V3-HLA-B35-10mers-161P2F10BEach peptide is a portion of SEQ ID NO: 7; eachstart position is specified, the length of peptideis 10 amino acids, and the end position for eachpeptide is the start position plus nine. Start Subsequence Score 8CPGGKPEALW 10 7 SCPGGKPEAL 1 1 RPTNVESCPG 0.6 6 ESCPGGKPEA 0.5 9PGGKPEALWV 0.03 10  GGKPEALWVE 0.03 3 TNVESCPGGK 0.02 4 NVESCPGGKP 0.0032 PTNVESCPGG 0.001 5 VESCPGGKPE 0.001 V4-HLA-B35-10mers-161P2F10BEach peptide is a portion of SEQ ID NO: 9; eachstart position is specified, the length of peptideis 10 amino acids, and the end position for eachpeptide is the start position plus nine. Start Subsequence Score 2TYLPTFETPI 0.04 1 KTYLPTFETP 0.02Tables XXII-XLIX:

TABLE XXII V1-HLA-A1-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 81; eachstart position is specified, the length of thepeptide is 9 amino acids and the end position foreach peptide is the start position plus 8. Pos 123456789 score 165SMDGFRAEY 29 431 KPDQHFKPY 28 442 PDLPKRLHY 26 188 KTCGIHSKY 24 618KNVDHCLLY 23 710 ITSNLVPMY 23 858 VSEILQLKT 23   8 ATEQPVKKN 21 193HSKYMRAMY 21 202 PTKTFPNHY 21 377 QTYCNKMEY 21 491 SMEAIFLAH 21 209HYTIVTGLY 19 391 PRINFFYMY 19 462 FVDQQWLAV 19 477 NCGGGNHGY 19 514NIEVYNLMC 19 638 MRMPMWSSY 19 772 TDVPIPTHY 19 859 SEILQLKTY 19  13VKKNTLKKY 18 389 YFPRINFFY 18 455 RIDKVHLFV 18 686 KNITHGFLY 18 737ATERNGVNV 18 745 VVSGPIFDY 18 843 ELLTGLDFY 18 152 CPEGFDLPP 17 167DGFRAEYLY 17 217 YPESHGIID 17 221 HGIIDNNMY 17 261 YQGLKAATY 17 281SFPSIYMPY 17 371 ADHGMDQTY 17 420 SEEIVRNLS 17 674 SESQKCSFY 17 698SNRTSDSQY 17 747 SGPIFDYNY 17 771 NTDVPIPTH 17 839 VRDVELLTG 17 115CSDDCLQKK 16 134 QGETSWLEE 16 253 QPMWLTAMY 16 312 ERPRFYTMY 16 381NKMEYMTDY 16 386 MTDYFPRIN 16 506 KTEVEPFEN 16 510 EPFENIEVY 16 540NHLLKVPFY 16 569 PTESLDCFC 16 602 ATVKVNLPF 16 619 NVDHCLLYH 16 622HCLLYHREY 16 651 LGDTSPLPP 16 659 PTVPDCLRA 16 673 PSESQKCSF 16 701TSDSQYDAL 16  89 CVESTRIWM 15 121 QKKDCCADY 15 210 YTIVTGLYP 15 278INGSFPSIY 15 309 PKAERPRFY 15 321 FEEPDSSGH 15 344 VVDHAFGML 15 419NSEEIVRNL 15 521 MCDLLRIQP 15 547 FYEPSHAEE 15 585 QLEQVNQML 15 719EEFRKMWDY 15 851 YQDKVQPVS 15  47 KLEKQGSCR 14 116 SDDCLQKKD 14 273GSEVAINGS 14 300 STLLKWLDL 14 508 EVEPFENIE 14 646 YTVPQLGDT 14 754NYDGHFDAP 14 847 GLDFYQDKV 14 V2-HLA-A1-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 82; eachstart position is specified, the length of thepeptide is 9 amino acids and the end position foreach peptide is the start position plus 8. Pos 123456789 score 8QRKDCCADY 15 2 CSDDCLQRK 14 3 SDDCLQRKD 14 9 RKDCCADYK 10 1 SCSDDCLQR  8V3-HLA-A1-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 85; eachstart position is specified, the length of thepeptide is 9 amino acids and the end position foreach peptide is the start position plus 8. Pos 123456789 score 5ESCPGGKPE 11 3 NVESCPGGK 10 9 GGKPEALWV 10 1 PTNVESCPG  6 4 VESCPGGKP  5V4-HLA-A1-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 88; eachstart position is specified, the length of thepeptide is 9 amino acids and the end position foreach peptide is the start position plus 8. Pos 123456789 score 1TYLPTFETP 7 2 YLPTFETPI 3

XXIII V1-HLA-A0201-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 81; eachstart position is specified, the length of thepeptide is 9 amino acids and the end position foreach peptide is the start position plus 8. Pos 123456789 score 179TLMPNINKL 30  40 GLGLGLRKL 29 723 KMWDYFHSV 25  27 VLLALLVIM 24  29LALLVIMSL 24  31 LLVIMSLGL 24 215 GLYPESHGI 24 277 AINGSFPSI 24 519NLMCDLLRI 24 572 SLDCFCPHL 24  33 VIMSLGLGL 23 360 NLHNCVNII 23 847GLDFYQDKV 23 867 YLPTFETTI 23  23 IACIVLLAL 22 298 RISTLLKWL 22 534GTHGSLNHL 22 607 NLPFGRPRV 22 768 HLANTDVPI 22  24 ACIVLLALL 21  26IVLLALLVI 21 223 IIDNNMYDV 21 259 AMYQGLKAA 21 460 HLFVDQQWL 21 592MLNLTQEEI 21 623 CLLYHREYV 21  21 YKIACIVLL 20  22 KIACIVLLA 20  25CIVLLALLV 20  65 GLENCRCDV 20 256 WLTAMYQGL 20 300 STLLKWLDL 20 337RVIKALQVV 20 385 YMTDYFPRI 20 439 YLTPDLPKR 20 537 GSLNHLLKV 20 560SVCGFANPL 20 600 ITATVKVNL 20 807 IIPHRPTNV 20 836 IARVRDVEL 20  28LLALLVIMS 19  37 LGLGLGLGL 19 204 KTFPNHYTI 19 451 AKNVRIDKV 19 455RIDKVHLFV 19 585 QLEQVNQML 19 653 DTSPLPPTV 19 709 LITSNLVPM 19 800WLDVLPFII 19   5 LTLATEQPV 18  36 SLGLGLGLG 18 285 IYMPYNGSV 18 351MLMEGLKQR 18 367 IILLADHGM 18 443 DLPKRLHYA 18 682 YLADKNITH 18 731VLLIKHATE 18 834 AHIARVRDV 18 840 RDVELLTGL 18 860 EILQLKTYL 18  35MSLGLGLGL 17  38 GLGLGLGLR 17 139 WLEENCDTA 17 165 SMDGFRAEY 17 173YLYTWDTLM 17 334 VSARVIKAL 17 397 YMYEGPAPR 17 440 LTPDLPKRL 17 462FVDQQWLAV 17 512 FENIEVYNL 17 538 SLNHLLKVP 17 565 ANPLPTESL 17 595LTQEEITAT 17 596 TQEEITATV 17 737 ATERNGVNV 17 738 TERNGVNVV 17 799GWLDVLPFI 17 825 ALWVEERFT 17 850 FYQDKVQPV 17 854 KVQPVSEIL 17 863QLKTYLPTF 17  34 IMSLGLGLG 16 157 DLPPVILFS 16 208 NHYTIVTGL 16 231VNLNKNFSL 16 301 TLLKWLDLP 16 330 AGGPVSARV 16 453 NVRIDKVHL 16 520LMCDLLRIQ 16 646 YTVPQLGDT 16 706 YDALITSNL 16 707 DALITSNLV 16 732LLIKHATER 16 774 VPIPTHYFV 16 796 NCPGWLDVL 16 806 FIIPHRPTN 16 837ARVRDVELL 16 861 ILQLKTYLP 16  18 LKKYKIACI 15 104 GETRLEASL 15 107RLEASLCSC 15 119 CLQKKDCCA 15 132 VCQGETSWL 15 211 TIVTGLYPE 15 305WLDLPKAER 15 328 GHAGGPVSA 15 340 KALQVVDHA 15 344 VVDHAFGML 15 369LLADHGMDQ 15 419 NSEEIVRNL 15 496 FLAHGPSFK 15 542 LLKVPFYEP 15 591QMLNLTQEE 15 594 NLTQEEITA 15 598 EEITATVKV 15 608 LPFGRPRVL 15 615VLQKNVDHC 15 692 FLYPPASNR 15 708 ALITSNLVP 15 735 KHATERNGV 15 761APDEITKHL 15 775 PIPTHYFVV 15 803 VLPFIIPHR 15 831 RFTAHIARV 15 843ELLTGLDFY 15 856 QPVSEILQL 15 865 KTYLPTFET 15  30 ALLVIMSLG 14  42GLGLRKLEK 14 150 SQCPEGFDL 14 155 GFDLPPVIL 14 191 GIHSKYMRA 14 268TYFWPGSEV 14 270 FWPGSEVAI 14 284 SIYMPYNGS 14 294 PFEERISTL 14 303LKWLDLPKA 14 336 ARVIKALQV 14 341 ALQVVDHAF 14 348 AFGMLMEGL 14 350GMLMEGLKQ 14 362 HNCVNIILL 14 368 ILLADHGMD 14 436 FKPYLTPDL 14 448LHYAKNVRI 14 524 LLRIQPAPN 14 548 YEPSHAEEV 14 581 QNSTQLEQV 14 588QVNQMLNLT 14 624 LLYHREYVS 14 640 MPMWSSYTV 14 643 WSSYTVPQL 14 656PLPPTVPDC 14 661 VPDCLRADV 14 664 CLRADVRVP 14 688 ITHGFLYPP 14 713NLVPMYEEF 14 716 PMYEEFRKM 14 730 SVLLIKHAT 14 742 GVNVVSGPI 14 764EITKHLANT 14 776 IPTHYFVVL 14 844 LLTGLDFYQ 14 853 DKVQPVSEI 14V2-HLA-A0201-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 82; eachstart position is specified, the length of thepeptide is 9 amino acids and the end position foreach peptide is the start position plus 8. Pos 123456789 score 6CLQRKDCCA 15  2 CSDDCLQRK 5 3 SDDCLQRKD 5 V3-HLA-A0201-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 85; eachstart position is specified, the length of thepeptide is 9 amino acids and the end position foreach peptide is the start position plus 8. Pos 123456789 score 7CPGGKPEAL 14 6 SCPGGKPEA 13 9 GGKPEALWV 13 V4-HLA-A0201-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 88; eachstart position is specified, the length of thepeptide is 9 amino acids and the end position foreach peptide is the start position plus 8. Pos 123456789 score 2YLPTFETPI 21

Table XXIV-V1-HLA-A0203-9mers-161P2F10B Pos 123456789 score No ResultsFound. Table XXIV-V2-HLA-A0203-9mers-161P2F10B Pos 123456789 score NoResults Found. Table XXIV-V3-HLA-A0203-9mers-161P2F10B Pos 123456789score No Results Found. Table XXIV-V4-HLA-A0203-9mers-161P2F10B Pos123456789 score No Results Found.

TABLE XXV V1-HLA-A3-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 81; eachstart position is specified, the length of thepeptide is 9 amino acids and the end position foreach peptide is the start position plus 8. Pos 123456789 score 670RVPPSESQK 33 496 FLAHGPSFK 27 692 FLYPPASNR 27  42 GLGLRKLEK 26 423IVRNLSCRK 26   6 TLATEQPVK 25 614 TVLQKNVDH 25  12 PVKKNTLKK 24 302LLKWLDLPK 24 610 FGRPRVLQK 24  47 KLEKQGSCR 23 708 ALITSNVLP 23  26IVLLALLVI 22 447 RLHYAKNVR 22 782 VVLTSCKNK 22 838 RVRDVELLT 22 337RVIKALQVV 21 405 RIRAHNIPH 21 469 AVRSKSNTN 21 597 QEEITATVK 21 628REYVSGFGK 21 682 YLADKNITH 21 731 VLLIKHATE 21 732 LLIKHATER 21 814NVESCPEGK 21 857 PVSEILQLK 21 863 QLKTYLPTF 21  39 LGLGLGLRK 20 351MLMEGLKQR 20 624 LLYHREYVS 20 843 ELLTGLDFY 20 196 YMRAMYPTK 19 227NMYDVNLNK 19 338 VIKALQVVD 19 341 ALQVVDHAF 19 544 KVPFYEPSH 19 664CLRADVRVP 19 802 DVLPFIIPH 19 806 FIIPHRPTN 19 827 WVEERFTAH 19  27VLLALLVIM 18  30 ALLVIMSLG 18  38 GLGLGLGLR 18 107 RLEASLCSC 18 114SCSDDCLQK 18 261 YQGLKAATY 18 275 EVAINGSFP 18 305 WLDLPKAER 18 368ILLADHGMD 18 422 EIVRNLSCR 18 453 NVRIDKVHL 18 524 LLRIQPAPN 18 550PSHAEEVSK 18 745 VVSGPIFDY 18 759 FDAPDEITK 18 835 HIARVRDVE 18 157DLPPVILFS 17 162 ILFSMDGFR 17 186 KLKTCGIHS 17 215 GLYPESHGI 17 296EERISTLLK 17 439 YLTPDLPKR 17 458 KVHLFVDQQ 17 619 NVDHCLLYH 17 647TVPQLGDTS 17 668 DVRVPPSES 17 713 NLVPMYEEF 17 854 KVQPVSEIL 17   7LATEQPVKK 16  44 GLRKLEKQG 16  68 NCRCDVACK 16  94 RIWMCNKFR 16 122KKDCCADYK 16 131 SVCQGETSW 16 343 QVVDHAFGM 16 355 GLKQRNLHN 16 369LLADHGMDQ 16 442 PDLPKRLHY 16 455 RIDKVHLFV 16 498 AHGPSFKEK 16 523DLLRIQPAP 16 526 RIQPAPNNG 16 560 SVCGFANPL 16 630 YVSGFGKAM 16 678KCSFYLADK 16 698 SNRTSDSQY 16 768 HLANTDVPI 16 V2-HLA-A3-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 82; eachstart position is specified, the length of thepeptide is 9 amino acids and the end position foreach peptide is the start position plus 8. Pos 123456789 score 9RKDCCADYK 17 6 CLQRKDCCA 14 8 QRKDCCADY 13 1 SCSDDCLQR 12 2 CSDDCLQRK 11V3-HLA-A3-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 85; eachstart position is specified, the length of thepeptide is 9 amino acids and the end position foreach peptide is the start position plus 8. Pos 123456789 score 3NVESCPGGK 22 9 GGKPEALWV 11 V4-HLA-A3-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 88; eachstart position is specified, the length of thepeptide is 9 amino acids and the end position foreach peptide is the start position plus 8. Pos 123456789 score 2YLPTFETPI 11

TABLE XXVI Pos 123456789 score V1-HLA-A26-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 81; each start positionis specified, the length of the peptide is 9 amino acids and the endposition for each peptide is the start position plus 8. 510 EPFENIEVY 31516 EVYNLMCDL 29 773 DVPIPTHYF 26 388 DYFPRINFF 25 719 EEFRKMWDY 25 422EIVRNLSCR 24 710 ITSNLVPMY 24 745 VVSGPIFDY 24 802 DVLPFIIPH 24 843ELLTGLDFY 24 275 EVAINGSFP 23 587 EQVNQMLNL 23 860 EILQLKTYL 23 167DGFRAEYLY 22 188 KTCGIHSKY 22 297 ERISTLLKW 21 312 ERPRFYTMY 21 508EVEPFENIE 21 602 ATVKVNLPF 21 10 EQPVKKNTL 20 344 VVDHAFGML 20 534GTHGSLNHL 20 555 EVSKFSVCG 20 841 DVELLTGLD 20 859 SEILQLKTY 20 32LVIMSLGLG 19 72 DVACKDRGD 19 105 ETRLEASLC 19 136 ETSWLEENC 19 172EYLYTWDTL 19 230 DVNLNKNFS 19 300 STLLKWLDL 19 377 QTYCNKMEY 19 488EFRSMEAIF 19 560 SVCGFANPL 19 599 EITATVKVN 19 653 DTSPLPPTV 19 668DVRVPPSES 19 764 EITKHLANT 19 840 RDVELLTGL 19 854 KVQPVSEIL 19 856QPVSEILQL 19 202 PTKTFPNHY 18 337 RVIKALQVV 18 364 CVNIILLAD 18 440LTPDLPKRL 18 453 NVRIDKVHL 18 454 VRIDKVHLF 18 646 YTVPQLGDT 18 675ESQKCSFYL 18 720 EFRKMWDYF 18 772 TDVPIPTHY 18 824 EALWVEERF 18 857PVSEILQLK 18 24 ACIVLLALL 17 77 DRGDCCWDF 17 178 DTLMPNINK 17 274SEVAINGSF 17 298 RISTLLKWL 17 551 SHAEEVSKF 17 554 EEVSKFSVC 17 629EYVSGFGKA 17 660 TVPDCLRAD 17 685 DKNITHGFL 17 726 DYFHSVLLI 17 853DKVQPVSEI 17 2 ESTLTLATE 16 21 YKIACIVLL 16 33 VIMSLGLGL 16 87 DTCVESTRI16 145 DTAQQSQCP 16 154 EGFDLPPVI 16 179 TLMPNINKL 16 219 ESHGIIDNN 16225 DNNMYDVNL 16 294 PFEERISTL 16 333 PVSARVIKA 16 419 NSEEIVRNL 16 421EEIVRNLSC 16 513 ENIEVYNLM 16 600 ITATVKVNL 16 603 TVKVNLPFG 16 618KNVDHCIIY 16 744 NVVSGPIFD 16 830 ERFTAHIAR 16 837 ARVRDVELL 16 13VKKNTLKKY 15 26 IVLLALLVI 15 204 KTFPNHYTI 15 221 HGIIDNNMY 15 343QVVDHAFGM 15 391 PRINFEYMY 15 458 KVHLFVDQQ 15 493 EAIFLAHGP 15 540NHLLKVPFY 15 568 LPTESLDCF 15 595 LTQEEITAT 15 598 EEITATVKV 15 619NVDHCLLYH 15 626 YHREYVSGF 15 686 KNITHGFLY 15 763 DEITKHLAN 15 781FVVLTSCKN 15 V2-HLA-A26-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 82; each start position is specified, the length of thepeptide is 9 amino acids and the end position for each peptide is thestart position plus 8. 8 QRKDCCADY 11 4 DDCLQRKDC 8 5 DCLQRKDCC 8 2CSDDCLQRK 5 V3-HLA-A26-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 85; each start position is specified, the length of thepeptide is 9 amino acids and the end position for each peptide is thestart position plus 8. 3 NVESCPGGK 12 5 ESCPGGKPE 12 7 CPGGKPEAL 10 1PTNVESCPG 8 2 TNVESCPGG 7 V4-HLA-A26-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 88; each start positionis specified, the length of the peptide is 9 amino acids and the endposition for each peptide is the start position plus 8. 1 TYLPTFETP 3

TABLE XXVII Pos 123456789 score V1-HLA-B0702-9mers-161P2F10B8Each peptide is a portion of SEQ ID NO: 81; each start positionis specified, the length of the peptide is 9 amino acids and the endposition for each peptide is the start position plus 8. 776 IPTHYFVVL 25761 APDEITKHL 24 608 LPFGRPRVL 23 818 CPEGKPEAL 23 856 QPVSEILQL 22 313RPRFYTMYF 20 403 APRIRAHNI 20 657 LPPTVPDCL 20 793 TPENCPGWL 20 287MPYNGSVPF 19 308 LPKAERPRF 19 390 FPRINFFYM 19 612 RPRVLQKNV 19 661VPDCLRADV 19 181 MPNINKLKT 18 206 FPNHYTIVT 18 655 SPLPPTVPD 18 774VPIPTHYFV 18 293 VPFEERIST 17 640 MPMWSSYTV 17 155 GFDLPPVIL 16 158LPPVILFSM 16 545 VPFYEPSHA 16 565 ANPLPTESL 16 568 LPTESLDCF 16 808IPHRPTNVE 16 35 MSLGLGLGL 15 37 LGLGLGLGL 15 152 CPEGFDLPP 15 836IARVRDVEL 15 23 IACIVLLAL 14 33 VIMSLGLGL 14 100 KFRCGETRL 14 323EPDSSGHAG 14 431 KPDQHFKPY 14 441 TPDLPKRLH 14 453 NVRIDKVHL 14 549EPSHAEEVS 14 600 ITATVKVNL 14 643 WSSYTVPQL 14 649 PQLGDTSPL 14 822KPEALWVEE 14 21 YKIACIVLL 13 24 ACIVLLALL 13 58 CFDASFRGL 13 132VCQGETSWL 13 201 YPTKTFPNH 13 253 QPMWLTAMY 13 271 WPGSEVAIN 13 282FPSIYMPYN 13 298 RISTLLKWL 13 332 GPVSARVIK 13 348 AFGMLMEGL 13 401GPAPRIRAH 13 411 IPHDFFSFN 13 489 FRSMEAIFL 13 530 APNNGTHGS 13 560SVCGFANPL 13 587 EQVNQMLNL 13 658 PPTVPDCLR 13 695 PPASNRTSD 13 796NCPGWLDVL 13 797 CPGWLDVLP 13 837 ARVRDVEII 13 1 MESTLTLAT 12 11QPVKKNTLK 12 20 KYKIACIVL 12 31 LLVIMSLGL 12 112 LCSCSDDCL 12 150SQCPEGFDL 12 179 TLMPNINKL 12 198 RAMYPTKTF 12 208 NHYTIVTGL 12 225DNNMYDVNL 12 249 WWHGQPMWL 12 277 AINGSFPSI 12 300 STLLKWLDL 12 311AERPRFYTM 12 334 VSARVIKAL 12 344 VVDHAFGML 12 398 MYEGPAPRI 12 400EGPAPRIRA 12 408 AHNIPHDFF 12 428 SCRKPDQHF 12 436 FKPYLTPDL 12 455RIDKVHLFV 12 500 GPSFKEKTE 12 510 EPEENIEVY 12 528 QPAPNNGTH 12 531PNNGTHGSL 12 534 GTHGSLNHL 12 572 SLDCFCPHL 12 602 ATVKVNLPF 12 630YVSGFGKAM 12 672 PPSESQKCS 12 675 ESQKCSFYL 12 701 TSDSQYDAL 12 706YDALITSNL 12 724 MWDYFHSVL 12 725 WDYFHSVLL 12 840 RDVELLTGL 12 854KVQPVSEIL 12 860 EILQLKTYL 12 V2-B0702-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 82; each start positionis specified, the length of the peptide is 9 amino acids and the endposition for each peptide is the start position plus 8. 6 CLQRKDCCA 6 1SCSDDCLQR 4 7 LQRKDCCAD 4 9 RKDCCADYK 2 2 CSDDCLQRK 1 3 SDDCLQRKD 1 4DDCLQRKDC 1 8 QRKDCCADY 1 V3-B0702-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 85; each start positionis specified, the length of the peptide is 9 amino acids and the endposition for each peptide is the start position plus 8. 7 CPGGKPEAL 24V4-B0702-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 88; each start position is specified, the length of thepeptide is 9 amino acids and the end position for each peptide is thestart position plus 8. 2 YLPTFETPI 8

TABLE XXVIII Pos 123456789 score V1-HLA-B08-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 81; each start positionis specified, the length of the peptide is 9 amino acids and the endposition for each peptide is the start position plus 8. 836 IARVRDVEL 3218 LKKYKIACI 28 184 INKLKTCGI 28 818 CPEGKPEAL 27 355 GLKQRNLHN 26 403APRIRAHNI 26 502 SFKEKTEVE 24 608 LPFGRPRVL 24 10 EQPVKKNTL 23 300STLLKWLDL 23 231 VNLNKNFSL 22 308 LPKAERPRF 22 786 SCKNKSHTP 22 863QLKTYLPTF 22 294 PFEERISTL 21 353 MEGLKQRNL 21 20 KYKIACIVL 20 616LQKNVDHCL 20 98 CNKFRCGET 19 761 APDEITKHL 19 40 GLGLGLRKL 18 53SCRKKCFDA 18 74 ACKDRGDCC 18 166 MDGFRAEYL 18 238 SLSSKEQNN 18 313RPRFYTMYF 18 448 LHYAKNVRI 18 454 VRIDKVHLF 18 460 HLFVDQQWL 18 500GPSFKEKTE 18 542 IIKVPFYEP 18 556 VSKFSVCGF 18 572 SLDCFCPHL 18 776IPTHYFVVL 18 861 ILQLKTYLP 18 11 QPVKKNTLK 17 15 KNTLKKYKI 17 17TLKKYKIAC 17 42 GLGLRKLEK 17 179 TLMPNINKL 17 263 GLKAATYFW 17 401GPAPRIRAH 17 444 LPKRLHYAK 17 453 NVRIDKVHL 17 504 KEKTEVEPF 17 585QLEQVNQML 17 682 YLADKNITH 17 731 VIIIKHATE 17 856 QPVSEILQL 17 23IACIVLLAL 16 29 LALLVIMSL 16 31 LLVIMSLGL 16 55 RKKCFDASF 16 100KFRCGETRL 16 119 CLQKKDCCA 16 126 CADYKSVCQ 16 186 KLKTCGIHS 16 256WLTAMYQGL 16 302 LLKWLDLPK 16 338 VIKALQVVD 16 467 WLAVRSKSN 16 469AVRSKSNTN 16 601 TATVKVNLP 16 610 FGRPRVLQK 16 657 LPPTVPDCL 16 684ADKNITHGF 16 736 HATERNGVN 16 793 TPENCPGWL 16 860 EILQLKTYL 16 33VIMSLGLGL 15 51 QGSCRKKCF 15 191 GIHSKYMRA 15 240 SSKEQNNPA 15 298RISTLLKWL 15 388 DYFPRINFF 15 626 YHREYVSGF 15 V2-B08-9mers-61P2F10BEach peptide is a portion of SEQ ID NO: 82; each start positionis specified, the length of the peptide is 9 amino acids and the endposition for each peptide is the start position plus 8. 6 CLQRKDCCA 16 8QRKDCCADY 10 5 DCLQRKDCC 8 7 LQRKDCCAD 7 V3-HLA-B08-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 85; each start positionis specified, the length of the peptide is 9 amino acids and the endposition for each peptide is the start position plus 8. 7 CPGGKPEAL 27V4-HLA-B08-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 88; each start position is specified, the length of thepeptide is 9 amino acids and the end position for each peptide is thestart position plus 8. 2 YLPTFETPI 12

TABLE XXIX Pos 123456789 score V1-HLA-B1510-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 81; each start positionis specified, the length of the peptide is 9 amino acids and the endposition for each peptide is the start position plus 8. 192 IHSKYMRAM 24578 PHLQNSTQL 22 208 NHYTIVTGL 21 535 THGSLNHLL 21 361 LHNCVNIIL 20 551SHAEEVSKF 19 626 YHREYVSGF 19 328 GHAGGPVSA 17 408 AHNIPHDFF 17 220SHGIIDNNM 16 600 ITATVKVNL 16 608 LPFGRPRVL 16 776 IPTHYFVVL 16 836IARVRDVEL 16 155 GFDLPPVIL 15 179 TLMPNINKL 15 419 NSEEIVRNL 15 809PHRPTNVES 15 10 EQPVKKNTL 14 21 YKIACIVLL 14 40 GLGLGLRKL 14 294PFEERISTL 14 440 LTPDLPKRL 14 448 LHYAKNVRI 14 757 GHFDAPDEI 14 818CPEGKPEAL 14 23 IACIVLLAL 13 100 KFRCGETRL 13 250 WHGQPMWLT 13 334VSARVIKAL 13 346 DHAFGMLME 13 362 HNCVNIILL 13 434 QHFKPYLTP 13 453NVRIDKVHL 13 498 AHGPSFKEK 13 643 WSSYTVPQL 13 767 KHLANTDVP 13 778THYFVVLTS 13 793 TPENCPGWL 13 796 NCPGWLDVL 13 834 AHIARVRDV 13 860EILQLKTYL 13 20 KYKIACIVL 12 58 CFDASFRGL 12 150 SQCPEGFDL 12 172EYLYTWDTL 12 225 DNNMYDVNL 12 249 WWHGQPMWL 12 353 MEGLKQRNL 12 432PDQHFKPYL 12 516 EVYNLMCDL 12 534 GTHGSLNHL 12 540 NHLLKVPFY 12 585QLEQVNQML 12 587 EQVNQMLNL 12 621 DHCLLYHRE 12 675 ESQKCSFYL 12 689THGFLYPPA 12 701 TSDSQYDAL 12 706 YDALITSNL 12 724 MWDYFHSVL 12 725WDYFHSVLL 12 728 FHSVLLIKH 12 735 KHATERNGV 12 791 SHTPENCPG 12 854KVQPVSEIL 12 24 ACIVLLALL 11 29 LALLVIMSL 11 35 MSLGLGLGL 11 37LGLGLGLGL 11 104 GETRLEASL 11 112 LCSCSDDCL 11 132 VCQGETSWL 11 295FEERISTLL 11 298 RISTLLKWL 11 348 AFGMLMEGL 11 436 FKPYLTPDL 11 460HLFVDQQWL 11 482 NHGYNNEFR 11 489 FRSMEAIFL 11 512 FENIEVYNL 11 531PNNGTHGSL 11 560 SVCGFANPL 11 565 ANPLPTESL 11 572 SLDCFCPHL 11 617QKNVDHCLL 11 649 PQLGDTSPL 11 657 LPPTVPDCL 11 761 APDEITKHL 11 837ARVRDVELL 11 840 RDVELLTGL 11 856 QPVSEILQL 11V2-HLA-B1510-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 82; each start position is specified, the length of thepeptide is 9 amino acids and the end position for each peptide is thestart position plus 8. 2 CSDDCLQRK 3 1 SCSDDCLQR 2 3 SDDCLQRKD 2 5DCLQRKDCC 2 7 LQRKDCCAD 2 4 DDCLQRKDC 1 8 QRKDCCADY 1V3-HLA-B1510-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 85; each start position is specified, the length of thepeptide is 9 amino acids and the end position for each peptide is thestart position plus 8. 7 CPGGKPEAL 13 V4-HLA-B1510-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 88; each start positionis specified, the length of the peptide is 9 amino acids and the endposition for each peptide is the start position plus 8. 1 TYLPTFETP 4 2YLPTFETPI 1

TABLE XXX Pos 123456789 score V1-HLA-B2705-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 81; each start positionis specified, the length of the peptide is 9 amino acids and the endposition for each peptide is the start position plus 8. 93 TRIVMCNKF 25454 VRIDKVHLF 24 830 ERFTAHIAR 24 429 CRKPDQHFK 23 489 FRSMEAIFL 23 837ARVRDVELL 23 77 DRGDCCWDF 21 391 PRINFFYMY 21 638 MRMPMWSSY 21 312ERPRFYTMY 20 721 FRKWMDYFH 20 840 RDVELLTGL 20 29 LALLVIMSL 19 40GLGLGLRKL 19 525 LRIQPAPNN 19 614 RVLQKNVDH 19 39 LGLGLGLRK 18 100KFRCGETRL 18 388 DYFPRINFF 18 470 VRSKSNTNC 18 481 GNHGYNNEF 18 534GTHGSLNHL 18 578 PHLQNSTQL 18 632 SGFGKAMRM 18 670 RVPPSESQK 18 692FLYPPASNR 18 732 LLIKHATER 18 860 EILQLKTYL 18 11 QPVKKNTLK 17 38GLGLGLGLR 17 42 GLGLRKLEK 17 48 LEKQGSCRK 17 55 RKKCFDASF 17 178DRLMPNINK 17 179 TLMPNINKL 17 227 NMYDVNLNK 17 262 QGLKAATYF 17 374GMDQTYCNK 17 446 KRLHYAKNV 17 447 RLHYAKNVR 17 602 ATVKVNLPF 17 611GRPRVLQKN 17 628 REYVSGFGK 17 856 QPVSEILQL 17 21 YKIACIVLL 16 24ACIVLLALL 16 35 MSLGLGLGL 16 37 LGLGLGLGL 16 104 GETRLEASL 16 155GFDLPPVIL 16 156 FDLPPVILF 16 187 LKTCGIHSK 16 188 KTCGIHSKY 16 198RAMYPTKTF 16 208 NHYTIVTGL 16 287 MPYNGSVPF 16 294 PFEERISTL 16 297ERISTLLKW 16 298 RISTLLKWL 16 336 ARVIKALQV 16 353 MEGLKQRNL 16 387TDYFPRINF 16 397 YMYEGPAPR 16 510 EPEENIEVY 16 551 SHAEEVSKF 16 600ITATVKVNL 16 608 LPFGRPRVL 16 739 ERNGVNVVS 16 760 DAPDEITKH 16 6TLATEQPVK 15 12 PVKKNTLKK 15 47 KLEKQGSCR 15 49 EKQGSCRKK 15 63FRGLENCRC 15 94 RIWMCNKFR 15 106 TRLEASLCS 15 162 ILFSMDGFR 15 190CGIHSKYMR 15 229 YDVNLNKNF 15 231 VNLNKNFSL 15 300 STLLKWLDL 15 305WLDLPKAER 15 313 RPRFYTMYF 15 329 HAGGPVSAR 15 332 GPVSARVIK 15 351MLMEGLKQR 15 359 RNLHNCVNI 15 401 GPAPRIRAH 15 406 IRAHNIPHD 15 407RAHNIPHDF 15 419 NSEEIVRNL 15 423 IVRNLSCRK 15 438 PYLTPDLPK 15 439YLTPDLPKR 15 460 HLFVDQQWL 15 463 VDQQWLAVR 15 495 IFLAHGPSF 15 512FENIEVYNL 15 613 PRVLQKNVD 15 649 PQLGDTSPL 15 669 VRVPPSESQ 15 706YDALITSNL 15 782 VVLTSCKNK 15 802 DVLPFIIPH 15 803 VLPFIIPHR 15 824EALWVEERF 15 854 KVQPVSEIL 15 7 LATEQPVKK 14  10 EQPVKKNTL 14  14KKNTLKKYK 14 15 KNTLKKYKI 14 20 KYKIACIVL 14 23 IACIVLLAL 14 56KKCFDASFR 14 62 SFRGLENCR 14 69 CRCDVACKD 14 70 RCDVACKDR 14 99NKFRCGETR 14 132 VCQGETSWL 14 169 FRAEYLYTW 14 197 MRAMYPTKT 14 204KTFPNHYTI 14 215 GLYPESHGI 14 225 DNNMYDVNL 14 234 NKNFSLSSK 14 252GQPMWLTAM 14 274 SEVAINGSF 14 295 FEERISTLL 14 308 LPKAERPRF 14 314PREYTMYFE 14  334 VSARVIKAL 14  362 HNCVNIILL 14 405 RIRAHNIPH 14 424VRNLSCRKP 14 428 SCRKPDQHF 14 452 KNVRIDKVH 14 498 AHGPSFKEK 14 504KEKTEVEPF 14 518 YNLMCDLLR 14 533 NGTHGSLNH 14 539 LNHLLKVPF 14 540NHLLKVPFY 14 560 SVCGFANPL 14 565 ANPLPTESL 14 584 TQLEQVNQM 14 585QLEQVNQML 14 587 EQVNQMLNL 14 597 QEEITATVK 14 610 FGRPRVLQK 14 631VSGFGKAMR 14 678 KCSFYLADK 14 716 PMYEEFRKM 14 725 WDYFHSVLL 14 745VVSGPIFDY 14 771 NTDVPIPTH 14 796 NCPGWLDVL 14 799 GWLDVLPFI 14 836IARVRDVEL 14 839 VRDVELLTG 14 842 VELLTGLDF 14 26 IVLLALLVI 13 27VLLALLVIM 13 31 LLVIMSLGL 13 45 LRKLEKQGS 13 86 EDTCVESTR 13 101FRCGETRLE 13 115 CSDDCLQKK 13 122 KKDCCADYK 13 161 VILFSMDGF 13 172EYLYTWDTL 13 173 YLYTWDTLM 13 201 YPTKTFPNH 13 220 SHGIIDNNM 13 249WWHGQPMWL 13 261 YQGLKAATY 13 291 GSVPFEERI 13 302 LLKWLDLPK 13 341ALQVVDHAF 13 354 EGLKQRNLH 13 358 QRNLHNCVN 13 361 LHNCVNIIL 13 365VNIILLADH 13 367 IILLADHGM 13 371 ADHGMDQTY 13 377 QTYCNKMEY 13 398MYEGPAPRI 13 404 PRIRAHNIP 13 417 SFNSEEIVR 13 422 EIVRNLSCR 13 440LTPDLPKRL 13 442 PDLPKRLHY 13 448 LHYAKNVRI 13 465 QQWLAVRSK 13 477NCGGGNHGY 13 484 GYNNEFRSM 13 496 FLAHGPSFK 13 513 ENIEVYNLM 13 516EVYNLMCDL 13 531 PNNGTHGSL 13 550 PSHAEEVSK 13 568 LPTESLDCF 13 606VNLPFGRPR 13 620 VDHCLLYHR 13 626 YHREYVSGF 13 643 WSSYTVPQL 13 662PDCLRADVR 13 675 ESQKCSFYL 13 713 NLVPMYEEF 13 715 VPMYEEFRK 13 719EEFRKMWDY 13 727 YFHSVLLIK 13 728 FHSVLLIKH 13 743 VNVVSGPIF 13 751FDYNYDGHF 13 757 GHFDAPDEI 13 759 FDAPDEITK 13 761 APDEITKHL 13 772TDVPIPTHY 13 776 IPTHYFVVL 13 780 YFVVLTSCK 13 798 PGWLDVLPF 13 818CPEGKPEAL 13 823 PEALWVEER 13 832 FTAHIARVR 13 843 ELLTGLDFY 13 853DKVQPVSEI 13 857 PVSEILQLK 13 859 SEILQLKTY 13 863 QLKTYLPTF 13 33VIMSLGLGL 12 54 CRKKCFDAS 12 68 NCRCDVACK 12 87 DTCVESTRI 12 92STRIWMCNK 12 112 LCSCSDDCL 12 114 SCSDDCLQK 12 148 QQSQCPEGF 12 150SQCPEGFDL 12 154 EGFDLPPVI 12 165 SMDGFRAEY 12 167 DGFRAEYLY 12 180LMPNINKLK 12 185 NKLKTCGIH 12 196 YMRAMYPTK 12 221 HGIIDNNMY 12 257LTAMYQGLK 12 277 AINGSFPSI 12 280 GSFPSIYMP 12 290 NGSVPFEER 12 296EERISTLLK 12 307 DLPKAERPR 12 311 AERPRFYTM 12 331 GGPVSARVI 12 339IKALQVVDH 12 348 AFGMLMEGL 12 382 KMEYMTDYF 12 408 AHNIPHDFF 12 431KPDQHFKPY 12 432 PDQHFKPYL 12 436 FKPYLTPDL 12 444 LPKRLHYAK 12 450YAKNVRIDK 12 453 NVRIDKVHL 12 475 NTNCGGGNH 12 535 THGSLNHLL 12 536HGSLNHLLK 12 544 KVPFYEPSH 12 556 VSKFSVCGF 12 604 VKVNLPFGR 12 616LQKNVDHCL 12 618 KNVDHCLLY 12 622 HCLLYHREY 12 627 HREYVSGEG 12 658PPTVPDCLR 12 665 LRADVRVPP 12 673 PSESQKCSF 12 674 SESQKCSFY 12 680SFYLADKNI 12 682 YLADKNITH 12 684 ADKNITHGF 12 686 KNITHGFLY 12 698SNRTSDSQY 12 710 ITSNLVPMY 12 720 EFRKMWDYF 12 724 MWDYFHSVL 12 726DYFHSVLLI 12 747 SGPIFDYNY 12 810 HRPTNVESC 12 846 TGLDFYQDK 12V2-HLA-B2705-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 82; each start position is specified, the length of thepeptide is 9 amino acids and the end position for each peptide is thestart position plus 8. 8 QRKDCCADY 19 9 RKDCCADYK 15 2 CSDDCLQRK 13 1SCSDDCLQR 12 V3-HLA-B2705-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 85; each start position is specified, the length of thepeptide is 9 amino acids and the end position for each peptide is thestart position plus 8. 7 CPGGKPEAL 13 3 NVESCPGGK 10 6 SCPGGKPEA 7V4-HLA-B2705-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 88; each start position is specified, the length of thepeptide is 9 amino acids and the end position for each peptide is thestart position plus 8. 2 YLPTFETPI 8 1 TYLPTFETP 4

TABLE XXXI Pos 123456789 score V1-HLA-B2709-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 81; each start positionis specified, the length of the peptide is 9 amino acids and the endposition for each peptide is the start position plus 8. 446 KRLHYAKNV 24837 ARVRDVELL 24 336 ARVIKALQV 23 489 FRSMEAIFL 21 454 VRIDKVHLF 20 93TRIWMCNKF 19 77 DRGDCCWDF 18 359 RNLHNCVNI 16 840 RDVELLTGL 15 19KKYKIACIV 14 104 GETRLEASL 14 155 GFDLPPVIL 14 204 KTFPNHYTI 14 208NHYTIVTGL 14 215 GLYPESHGI 14 291 GSVPFEERI 14 298 RISTLLKWL 14 525LRIQPAPNN 14 537 GSLNHLLKV 14 799 GWLDVLPFI 14 831 RFTAHIARV 14 856QPVSEILQL 14 35 MSLGLGLGL 13 37 LGLGLGLGL 13 40 GLGLGLRKL 13 100KFRCGETRL 13 106 TRLEASLCS 13 297 ERISTLLKW 13 300 STLLKWLDL 13 314PREYTMYFE 13 337 RVIKALQVV 13 407 RAHNIPHDF 13 419 NSEEIVRNL 13 455RIDKVHLFV 13 460 HLFVDQQWL 13 512 FENIEVYNL 13 534 GTHGSLNHL 13 578PHLQNSTQL 13 587 EQVNQMLNL 13 600 ITATVKVNL 13 611 GRPRVLQKN 13 612RPRVLQKNV 13 643 WSSYTVPQL 13 649 PQLGDTSPL 13 699 NRTSDSQYD 13 725WDYFHSVLL 13 757 GHFDAPDEI 13 776 IPTHYFVVL 13 854 KVQPVSEIL 13 15KNTLKKYKI 12 20 KYKIACIVL 12 21 YKIACIVLL 12 23 IACIVLLAL 12 24ACIVLLALL 12 26 IVLLALLVI 12 29 LALLVIMSL 12 31 LLVIMSLGL 12 33VIMSLGLGL 12 55 RKKCFDASF 12 156 FDLPPVILF 12 172 EYLYTWDTL 12 198RAMYPTKTF 12 225 DNNMYDVNL 12 231 VNLNKNFSL 12 313 RPRFYTMYF 12 331GGPVSARVI 12 391 PRINFFYMY 12 406 IRAHNIPHD 12 429 CRKPDQHFK 12 448LHYAKNVRI 12 453 NVRIDKVHL 12 495 IFLAHGPSF 12 516 EVYNLMCDL 12 584TQLEQVNQM 12 608 LPFGRPRVL 12 632 SGFGKAMRM 12 663 DCLRADVRV 12 669VRVPPSESQ 12 706 YDALITSNL 12 726 DYFHSVLLI 12 761 APDEITKHL 12 830ERETAHIAR 12 836 IARVRDVEL 12 860 EILQLKTYL 12 25 CIVLLALLV 11 27VLLALLVIM 11 63 ERGLENCRC 11 65 GLENCRCDV 11 69 CRCDVACKD 11 150SQCPEGFDL 11 154 EGFDLPPVI 11 166 MDGFRAEYL 11 169 FRAEYLYTW 11 179TLMPNINKL 11 252 GQPMWLTAM 11 256 WLTAMYQGL 11 262 QGLKAATYF 11 287MPYNGSVPF 11 295 FEERISTLL 11 311 AERPRFYTM 11 330 AGGPVSARV 11 348AFGMLMEGL 11 353 MEGLKQRNL 11 367 IILLADHGM 11 385 YMTDYFPRI 11 387TDYFPRINF 11 388 DYFPRINFF 11 398 MYEGPAPRI 11 403 APRIRAHNI 11 404PRIRAHNIP 11 432 PDQHFKPYL 11 436 FKPYLTPDL 11 440 LTPDLPKRL 11 470VRSKSNTNC 11 481 GNHGYNNEF 11 484 GYNNEFRSM 11 504 KEKTEVEPF 11 519NLMCDLLRI 11 535 THGSLNHLL 11 560 SVCGFANPL 11 565 ANPLPTESL 11 572SLDCFCPHL 11 602 ATVKVNLPF 11 613 PRVLQKNVD 11 617 QKNVDHCLL 11 638MRMPMWSSY 11 665 LRADVRVPP 11 680 SFYLADKNI 11 701 TSDSQYDAL 11 709LITSNLVPM 11 721 FRKMWDYFH 11 737 ATERNGVNV 11 739 ERNGVNVVS 11 742GVNVVSGPI 11 774 VPIPTHYFV 11 798 PGWLDVLPF 11 810 HRPTNVESC 11 820EGKPEALWV 11 824 EALWVEERF 11 834 AHIARVRDV 11 839 VRDVELLTG 11 842VELLTGLDF 11 847 GLDFYQDKV 11 V2-HLA-B2709-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 82; each start positionis specified, the length of the peptide is 9 amino acids and the endposition for each peptide is the start position plus 8. 8 QRKDCCADY 10 9RKDCCADYK 5 V3-HLA-B2709-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 85; each start position is specified, the length of thepeptide is 9 amino acids and the end position for each peptide is thestart position plus 8. 9 GGKPEALWV 14 7 CPGGKPEAL 10V4-HLA-B2709-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 88; each start position is specified, the length of thepeptide is 9 amino acids and the end position for each peptide is thestart position plus 8. 2 YLPTFETPI 8 1 TYLPTFETP 3

TABLE XXXII Pos 123456789 score V1-HLA-B4402-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 81; each start positionis specified, the length of the peptide is 9 amino acids and the endposition for each peptide is the start position plus 8. 859 SEILQLKTY 29719 EEFRKMWDY 25 242 KEQNNPAWW 24 487 NEFRSMEAI 24 295 FEERISTLL 23 504KEKTEVEPF 23 842 VELLTGLDF 23 274 SEVAINGSF 22 353 MEGLKQRNL 22 674SESQKCSFY 22 104 GETRLEASL 21 512 FENIEVYNL 21 819 PEGKPEALW 21 179TLMPNINKL 20 388 DYFPRINFF 20 828 VEERETAHI 20 311 AERPRFYTM 19 507TEVEPEENI 19 510 EPEENIEVY 19 598 EEITATVKV 19 21 YKIACIVLL 18 24ACIVLLALL 18 297 ERISTLLKW 18 334 VSARVIKAL 18 761 APDEITKHL 18 156FDLPPVILF 17 421 EEIVRNLSC 17 454 VRIDKVHLF 17 608 LPFGRPRVL 17 684ADKNITHGF 17 763 DEITKHLAN 17 1 MESTLTLAT 16 90 VESTRIWMC 16 204KTFPNHYTI 16 341 ALQVVDHAF 16 362 HNCVNIILL 16 442 PDLPKRLHY 16 565ANPLPTESL 16 686 KNITHGFLY 16 837 ARVRDVELL 16 9 TEQPVKKNT 15 10EQPVKKNTL 15 13 VKKNTLKKY 15 29 LALLVIMSL 15 93 TRIWMCNKF 15 154EGFDLPPVI 15 171 AEYLYTWDT 15 198 RAMYPTKTF 15 218 PESHGIIDN 15 294PFEERISTL 15 296 EERISTLLK 15 298 RISTLLKWL 15 371 ADHGMDQTY 15 391PRINFFYMY 15 399 YEGPAPRIR 15 431 KPDQHFKPY 15 440 LTPDLPKRL 15 745VVSGPIFDY 15 772 TDVPIPTHY 15 796 NCPGWLDVL 15 843 ELLTGLDFY 15 856QPVSEILQL 15 23 IACIVLLAL 14 40 GLGLGLRKL 14 51 QGSCRKKCF 14 141EENCDTAQQ 14 150 SQCPEGFDL 14 155 GFDLPPVIL 14 165 SMDGFRAEY 14 167DGFRAEYLY 14 172 EYLYTWDTL 14 188 KTCGIHSKY 14 208 NHYTIVTGL 14 221HGIIDNNMY 14 241 SKEQNNPAW 14 300 STLKVVLDL 14 312 ERPREYTMY 14 322EEPDSSGHA 14 403 APRIRAHNI 14 407 RAHNIPHDF 14 419 NSEEIVRNL 14 420SEEIVRNLS 14 540 NHLLKVPFY 14 554 EEVSKFSVC 14 560 SVCGFANPL 14 587EQVNQMLNL 14 602 ATVKVNLPF 14 618 KNVDHCLLY 14 638 MRMPMWSSY 14 713NLVPMYEEF 14 738 TERNGVNVV 14 818 CPEGKPEAL 14 829 EERFTAHIA 14 854KVQPVSEIL 14 V2-HLA-B4402-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 82; each start position is specified, the length of thepeptide is 9 amino acids and the end position for each peptide is thestart position plus 8. 8 QRKDCCADY 11 1 SCSDDCLQR 5V3-HLA-B4402-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 85; each start position is specified, the length of thepeptide is 9 amino acids and the end position for each peptide is thestart position plus 8. 7 CPGGKPEAL 14 4 VESCPGGKP 12 8 PGGKPEALW 11 5ESCPGGKPE 7 V4-HLA-B4402-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 88; each start position is specified, the length of thepeptide is 9 amino acids and the end position for each peptide is thestart position plus 8. 2 YLPTFETPI 10 1 TYLPTFETP 6

TABLE XXXIIII Pos 123456789 score V1-HLA-B5101-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 81; each start positionis specified, the length of the peptide is 9 amino acids and the endposition for each peptide is the start position plus 8. 707 DALITSNLV 26608 LPFGRPRVL 25 776 IPTHYFVVL 24 29 LALLVIMSL 23 331 GGPVSARVI 23 657LPPTVPDCL 23 23 IACIVLLAL 22 154 EGFDLPPVI 22 640 MPMWSSYTV 22 403APRIRAHNI 21 448 LHYAKNVRI 21 760 DAPDEITKH 20 761 APDEITKHL 20 774VPIPTHYFV 20 26 IVLLALLVI 19 612 RPRVLQKNV 19 661 VPDCLRADV 19 694YPPASNRTS 19 726 DYFHSVLLI 19 818 CPEGKPEAL 19 836 IARVRDVEL 19 7LATEQPVKK 18 37 LGLGLGLGL 18 793 TPENCPGWL 18 856 QPVSEILQL 18 330AGGPVSARV 17 340 KALQVVDHA 17 510 EPEENIEVY 17 867 YLPTFETTI 17 87DTCVESTRI 16 206 FPNHYTIVT 16 216 LYPESHGII 16 287 MPYNGSVPF 16 385YMTDYFPRI 16 437 KPYLTPDLP 16 568 LPTESLDCF 16 663 DCLRADVRV 16 683LADKNITHG 16 736 HATERNGVN 16 799 GWLDVLPFI 16 808 IPHRPTNVE 16 820EGKPEALWV 16 853 DKVQPVSEI 16 60 DASFRGLEN 15 201 YPTKTFPNH 15 208NHYTIVTGL 15 270 FWPGSEVAI 15 293 VPFEERIST 15 450 YAKNVRIDK 15 497LAHGPSFKE 15 552 HAEEVSKFS 15 601 TATVKVNLP 15 653 DTSPLPPTV 15 680SFYLADKNI 15 738 TERNGVNVV 15 769 LANTDVPIP 15 804 LPFIIPHRP 15 824EALWVEERF 15 5 LTLATEQPV 14 11 QPVKKNTLK 14 18 LKKYKIACI 14 19 KKYKIACIV14 43 LGLRKLEKQ 14 124 DCCADYKSV 14 158 LPPVILFSM 14 181 MPNINKLKT 14215 GLYPESHGI 14 258 TAMYQGLKA 14 308 LPKAERPRF 14 347 HAFGMLMEG 14 360NLHNCVNII 14 398 MYEGPAPRI 14 411 IPHDFFSFN 14 500 GPSFKEKTE 14 507TEVEPEENI 14 519 NLMCDLLRI 14 537 GSLNHLLKV 14 545 VPFYEPSHA 14 566NPLPTESLD 14 671 VPPSESQKC 14 672 PPSESQKCS 14 15 KNTLKKYKI 13 39LGLGLGLRK 13 184 INKLKTCGI 13 198 RAMYPTKTF 13 204 KTFPNHYTI 13 217YPESHGIID 13 265 KAATYFWPG 13 277 AINGSFPSI 13 329 HAGGPVSAR 13 337RVIKALQVV 13 359 RNLHNCVNI 13 440 LTPDLPKRL 13 446 KRLHYAKNV 13 468LAVRSKSNT 13 487 NEFRSMEAI 13 528 QPAPNNGTH 13 529 PAPNNGTHG 13 549EPSHAEEVS 13 564 FANPLPTES 13 577 CPHLQNSTQ 13 596 TQEEITATV 13 655SPLPPTVPD 13 757 GHFDAPDEI 13 775 PIPTHYFVV 13 796 NCPGWLDVL 13 807IIPHRPTNV 13 833 TAHIARVRD 13 850 FYQDKVQPV 13 41 LGLGLRKLE 12 73VACKDRGDC 12 126 CADYKSVCQ 12 146 TAQQSQCPE 12 152 CPEGFDLPP 12 176TWDTLMPNI 12 225 DNNMYDVNL 12 271 WPGSEVAIN 12 272 PGSEVAING 12 282FPSIYMPYN 12 285 IYMPYNGSV 12 361 LHNCVNIIL 12 370 LADHGMDQT 12 390FPRINFFYM 12 401 GPAPRIRAH 12 402 PAPRIRAHN 12 419 NSEEIVRNL 12 431KPDQHFKPY 12 441 TPDLPKRLH 12 444 LPKRLHYAK 12 455 RIDKVHLFV 12 509VEPFENIEV 12 548 YEPSHAEEV 12 592 MLNLTQEEI 12 598 EEITATVKV 12 600ITATVKVNL 12 636 KAMRMPMWS 12 648 VPQLGDTSP 12 651 LGDTSPLPP 12 666RADVRVPPS 12 715 VPMYEEFRK 12 723 KMWDYFHSV 12 742 GVNVVSGPI 12 768HLANTDVPI 12 778 THYFVVLTS 12 811 RPTNVESCP 12 822 KPEALWVEE 12 828VEERETAHI 12 834 AHIARVRDV 12 V2-HLA-B5101-9mers-161P2F10BEach peptide is a portion of SEQ ID NO: 82; each start positionis specified, the length of the peptide is 9 amino acids and the endposition for each peptide is the start position plus 8. 5 DCLQRKDCC 6 4DDCLQRKDC 5 2 CSDDCLQRK 3 3 SDDCLQRKD 3 7 LQRKDCCAD 3 1 SCSDDCLQR 2V3-HLA-B5101-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 85; each start position is specified, the length of thepeptide is 9 amino acids and the end position for each peptide is thestart position plus 8. 7 CPGGKPEAL 19 9 GGKPEALWV 16V4-HLA-B5101-9mers-161P2F10B Each peptide is a portion ofSEQ ID NO: 88; each start position is specified, the length of thepeptide is 9 amino acids and the end position for each peptide is thestart position plus 8. 2 YLPTFETPI 15 1 TYLPTFETP 7

TABLE XXXIV Pos 1234567890 score V1-HLA-A1-10MERS-161P2F10BEach peptide is a portion of SEQ ID NO: 81; each start positionis specified, the length of the peptide is 10 amino acids and the endposition for each peptide is the start position plus 9. 441 TPDLPKRLHY32 771 NTDVPIPTHY 31 858 VSEILQLKTY 30 673 PSESQKCSFY 29 718 YEEFRKMWDY27 370 LADHGMDQTY 25 746 VSGPIFDYNY 24 280 GSFPSIYMPY 23 617 QKNVDHCLLY23 164 FSMDGFRAEY 21 311 AERPREYTMY 21 386 MTDYFPRINF 21 388 DYFPRINFFY20 390 FPRINFFYMY 20 697 ASNRTSDSQY 20 220 SHGIIDNNMY 19 295 FEERISTLLK19 420 SEEIVRNLSC 19 506 KTEVEPFENI 19 508 EVEPFENIEV 19 737 ATERNGVNVV19 430 RKPDQHFKPY 18 685 DKNITHGFLY 18 800 WLDVLPFIIP 18 8 ATEQPVKKNT 1712 PVKKNTLKKY 17 155 GFDLPPVILF 17 166 MDGFRAEYLY 17 187 LKTCGIHSKY 17192 IHSKYMRAMY 17 201 YPTKTFPNHY 17 208 NHYTIVTGLY 17 277 AINGSFPSIY 17419 NSEEIVRNLS 17 476 TNCGGGNHGY 17 509 VEPEENIEVY 17 709 LITSNLVPMY 17841 DVELLTGLDF 17 115 CSDDCLQKKD 16 217 YPESHGIIDN 16 252 GQPMWLTAMY 16260 MYQGLKAATY 16 539 LNHLLKVPFY 16 569 PTESLDCFCP 16 637 AMRMPMWSSY 16701 TSDSQYDALI 16 762 PDEITKHLAN 16 842 VELLTGLDFY 16 120 LQKKDCCADY 15257 LTAMYQGLKA 15 273 GSEVAINGSF 15 308 LPKAERPRFY 15 321 FEEPDSSGHA 15376 DQTYCNKMEY 15 380 CNKMEYMTDY 15 547 FYEPSHAEEV 15 553 AEEVSKFSVC 15572 SLDCFCPHLQ 15 621 DHCLLYHREY 15 744 NVVSGPIFDY 15V2-HLA-A1-10MERS-161P2F10B Each peptide is a portion ofSEQ ID NO: 83; each start position is specified, the length of thepeptide is 10 amino acids and the end position for each peptide is thestart position plus 9. 3 CSDDCLQRKD 16 8 LQRKDCCADY 15 4 SDDCLQRKDC 1310 RKDCCADYKS 12 1 CSCSDDCLQR 10 V3-HLA-A1-10MERS-161P2F10BEach peptide is a portion of SEQ ID NO: 86; each start positionis specified, the length of the peptide is 10 amino acids and the endposition for each peptide is the start position plus 9. 4 NVESCPGGKP 136 ESCPGGKPEA 8 2 PTNVESCPGG 6 9 PGGKPEALWV 6 V4-HLA-A1-10MERS-161P2F10BEach peptide is a portion of SEQ ID NO: 89; each start positionis specified, the length of the peptide is 10 amino acids and the endposition for each peptide is the start position plus 9. 1 KTYLPTFETP 9 2TYLPTFETPI 4

TABLE XXXV Pos 1234567890 score V1-HLA-A0201-10mers-161P2F10BEach peptide is a portion of SEQ ID NO: 81; each start positionis specified, the length of the peptide is 10 amino acids and the endposition for each peptide is the start position plus 9. 28 LLALLVIMSL 3022 KIACIVLLAL 27 30 ALLVIMSLGL 26 36 SLGLGLGLGL 24 708 ALITSNLVPM 24 835HIARVRDVEL 24 17 TLKKYKIACI 23 222 GIIDNNMYDV 23 284 SIYMPYNGSV 23 806FIIPHRPTNV 23 23 IACIVLLALL 22 34 IMSLGLGLGL 22 39 LGLGLGLRKL 22 111SLCSCSDDCL 22 165 SMDGFRAEYL 22 178 DTLMPNINKL 22 215 GLYPESHGII 22 302LLKWLDLPKA 22 397 YMYEGPAPRI 22 439 YLTPDLPKRL 22 595 LTQEEITATV 22 615VLQKNVDHCL 22 25 CIVLLALLVI 21 157 DLPPVILFSM 21 352 LMEGLKQRNL 21 450YAKNVRIDKV 21 639 RMPMWSSYTV 21 723 KMWDYFHSVL 21 32 LVIMSLGLGL 20 293VPFEERISTL 20 360 NLHNCVNIIL 20 564 FANPLPTESL 20 591 QMLNLTQEEI 20 737ATERNGVNVV 20 836 IARVRDVELL 20 4 TLTLATEQPV 19 369 LLADHGMDQT 19 447RLHYAKNVRI 19 607 NLPFGRPRVL 19 656 PLPPTVPDCL 19 682 YLADKNITHG 19 692FLYPPASNRT 19 825 ALWVEERFTA 19 26 IVLLALLVIM 18 183 NINKLKTCGI 18 204KTFPNHYTIV 18 259 AMYQGLKAAT 18 267 ATYFWPGSEV 18 276 VAINGSFPSI 18 329HAGGPVSARV 18 347 HAFGMLMEGL 18 534 GTHGSLNHLL 18 594 NLTQEEITAT 18 833TAHIARVRDV 18 27 VLLALLVIMS 17 42 GLGLRKLEKQ 17 162 ILFSMDGFRA 17 175YTWDTLMPNI 17 180 LMPNINKLKT 17 186 KLKTCGIHSK 17 227 NMYDVNLNKN 17 335SARVIKALQV 17 361 LHNCVNIILL 17 418 FNSEEIVRNL 17 518 YNLMCDLLRI 17 536HGSLNHLLKV 17 552 HAEEVSKFSV 17 580 LQNSTQLEQV 17 624 LLYHREYVSG 17 660TVPDCLRADV 17 736 HATERNGVNV 17 768 HLANTDVPIP 17 775 PIPTHYFVVL 17 861ILQLKTYLPT 17 6 TLATEQPVKK 16 20 KYKIACIVLL 16 31 LLVIMSLGLG 16 131SVCQGETSWL 16 152 CPEGFDLPPV 16 196 YMRAMYPTKT 16 230 DVNLNKNFSL 16 269YFWPGSEVAI 16 336 ARKIKALQVV 16 366 NIILLADHGM 16 374 GMDQTYCNKM 16 467WLAVRSKSNT 16 526 RIQPAPNNGT 16 599 EITATVKVNL 16 606 VNLPFGRPRV 16 700RTSDSQYDAL 16 792 HTPENCPGWL 16 839 VRDVELLTGL 16 855 VQPVSEILQL 16 9TEQPVKKNTL 15 24 ACIVLLALLV 15 171 AEYLYTWDTL 15 224 IDNNMYDVNL 15 257LTAMYQGLKA 15 333 PVSARVIKAL 15 343 QVVDHAFGML 15 355 GLKQRNLHNC 15 359RNLHNCVNII 15 454 VRIDKVHLFV 15 460 HLFVDQQWLA 15 461 LFVDQQWLAV 15 491SMEAIFLAHG 15 496 FLAHGPSFKE 15 519 NLMCDLLRIQ 15 530 APNNGTHGSL 15 538SLNHLLKVPF 15 541 HLLKVPFYEP 15 583 STQLEQVNQM 15 592 MLNLTQEEIT 15 642MWSSYTVPQL 15 706 YDALITSNLV 15 731 VLLIKHATER 15 765 ITKHLANTDV 15 774VPIPTHYFVV 15 817 SCPEGKPEAL 15 827 WVEERFTAHI 15 846 TGLDFYQDKV 15 33VIMSLGLGLG 14 57 KCFDASFRGL 14 64 RGLENCRCDV 14 65 GLENCRCDVA 14 173YLYTWDTLMP 14 191 GIHSKYMRAM 14 232 NLNKNFSLSS 14 338 VIKALQVVDH 14 341ALQVVDHAFG 14 350 GMLMEGLKQR 14 368 ILLADHGMDQ 14 494 AIFLAHGPSF 14 508EVEPFENIEV 14 533 NGTHGSLNHL 14 547 FYEPSHAEEV 14 584 TQLEQVNQML 14 709LITSNLVPMY 14 722 RKMWDYFHSV 14 732 LLIKHATERN 14 734 IKHATERNGV 14 773DVPIPTHYFV 14 830 ERFTAHIARV 14 844 LLTGLDFYQD 14 849 DFYQDKVQPV 14 859SEILQLKTYL 14 866 TYLPTFETTI 14 V2-HLA-A0201-10mers-161P2F10BEach peptide is a portion of SEQ ID NO: 83; each start positionis specified, the length of the peptide is 10 amino acids and the endposition for each peptide is the start position plus 9. 7 CLQRKDCCAD 112 SCSDDCLQRK 7 6 DCLQRKDCCA 5 V3-HLA-A0201-10mers-161P2F10BEach peptide is a portion of SEQ ID NO: 86; each start positionis specified, the length of the peptide is 10 amino acids and the endposition for each peptide is the start position plus 9. 7 SCPGGKPEAL 159 PGGKPEALWV 8 V4-HLA-A0201-10mers-161P2F10BEach peptide is a portion of SEQ ID NO: 89; each start positionis specified, the length of the peptide is 10 amino acids and the endposition for each peptide is the start position plus 9. 2 TYLPTFETPI 121 KTYLPTFETP 8

TABLE XXXVI V1-HLA-A0203- 10mers-161P2F10BEach peptide is a portion of SEQ ID NO: 81; each start position isspecified, the length of the peptide is 10 amino acids and the endposition for each peptide is the start position plus 9. Pos 1234567890score 258 TAMYQGLKAA 19 259 AMYQGLKAAT 17 15 KNTLKKYKIA 10 21 YKIACIVLLA10 52 GSCRKKCFDA 10 65 GLENCRCDVA 10 101 FRCGETRLEA 10 118 DCLQKKDCCA 10138 SWLEENCDTA 10 162 ILFSMDGFRA 10 190 CGIHSKYMRA 10 239 LSSKEQNNPA 10250 WHGQPMWLTA 10 257 LTAMYQGLKA 10 268 TYFWPGSEVA 10 302 LLKWLDLPKA 10321 FEEPDSSGHA 10 327 SGHAGGPVSA 10 332 GPVSARVIKA 10 339 IKALQVVDHA 10362 HNCVNIILLA 10 394 NFFYMYEGPA 10 399 YEGPAPRIRA 10 442 PDLPKRLHYA 10460 HLFVDQQWLA 10 485 YNNEFRSMEA 10 489 FRSMEAIFLA 10 521 MCDLLRIQPA 10544 KVPFYEPSHA 10 556 VSKFSVCGFA 10 593 LNLTQEEITA 10 628 REYVSGFGKA 10658 PPTVPDCLRA 10 675 ESQKCSFYLA 10 688 ITHGFLYPPA 10 699 NRTSDSQYDA 10728 FHSVLLIKHA 10 752 DYNYDGHFDA 10 761 APDEITKHLA 10 816 ESCPEGKPEA 10825 ALWVEERFTA 10 828 VEERFTAHIA 10 16 NTLKKYKIAC 9 22 KIACIVLLAL 9 53SCRKKCFDAS 9 66 LENCRCDVAC 9 102 RCGETRLEAS 9 119 CLQKKDCCAD 9 139WLEENCDTAQ 9 163 LFSMDGFRAE 9 191 GIHSKYMRAM 9 240 SSKEQNNPAW 9 251HGQPMWLTAM 9 269 YFWPGSEVAI 9 303 LKWLDLPKAE 9 322 EEPDSSGHAG 9 328GHAGGPVSAR 9 333 PVSARVIKAL 9 340 KALQVVDHAF 9 363 NCVNIILLAD 9 395FFYMYEGPAP 9 400 EGPAPRIRAH 9 443 DLPKRLHYAK 9 461 LFVDQQWLAV 9 486NNEFRSMEAI 9 490 RSMEAIFLAH 9 522 CDLLRIQPAP 9 545 VPFYEPSHAE 9 557SKFSVCGFAN 9 594 NLTQEEITAT 9 629 EYVSGFGKAM 9 659 PTVPDCLRAD 9 676SQKCSFYLAD 9 689 THGFLYPPAS 9 700 RTSDSQYDAL 9 729 HSVLLIKHAT 9 753YNYDGHFDAP 9 762 PDEITKHLAN 9 817 SCPEGKPEAL 9 826 LWVEERFTAH 9 829EERFTAHIAR 9 V2-HLA-A0203- 10mers-161P2F10BEach peptide is a portion of SEQ ID NO: 83; each start position isspecified, the length of the peptide is 10 amino acids and the endposition for each peptide is the start position plus 9. Pos 1234567890score 6 DCLQRKDCCA 10 7 CLQRKDCCAD 9 8 LQRKDCCADY 8 V3-HLA-A0203-10mers-161P2F10B Each peptide is a portion of SEQ IDNO: 86; each start position is specified, the length of the peptideis 10 amino acids and the end position for each peptide is thestart position plus 9. Pos 1234567890 score 6 ESCPGGKPEA 10 7 SCPGGKPEAL9 8 CPGGKPEALW 8 V4-HLA-A0203- 10mers-161P2F10B Pos 1234567890 scoreNo Results Found.

TABLE XXXVII V1-HLA-A3-10mers- 161P2F10BEach peptide is a portion of SEQ ID NO: 81; each start position isspecified, the length of the peptide is 10 amino acids and the endposition for each peptide is the start position plus 9. Pos 1234567890score 38 GLGLGLGLRK 27 6 TLATEQPVKK 26 186 KLKTCGIHSK 26 301 TLLKWLDLPK25 337 RVIKALQVVD 25 838 RVRDVELLTG 25 47 KLEKQGSCRK 23 179 TLMPNINKLK23 256 WLTAMYQGLK 23 277 AINGSFPSIY 23 212 IVTGLYPESH 21 422 EIVRNLSCRK21 437 KPYLTPDLPK 21 443 DLPKRLHYAK 21 462 FVDQQWLAVR 21 494 AIFLAHGPSF21 605 KVNLPFGRPR 21 730 SVLLIKHATE 21 731 VLLIKHATER 21 781 FVVLTSCKNK21 841 DVELLTGLDF 21 195 KYMRAMYPTK 20 368 ILLADHGMDQ 20 495 IFLAHGPSFK20 624 LLYHREYVSG 20 630 YVSGFGKAMR 20 664 CLRADVRVPP 20 708 ALITSNLVPM20 714 LVPMYEEFRK 20 26 IVLLALLVIM 19 32 LVIMSLGLGL 19 107 RLEASLCSCS 19338 VIKALQVVDH 19 523 DLLRIQPAPN 19 538 SLNHLLKVPF 19 588 QVNQMLNLTQ 19596 TQEEITATVK 19 609 PFGRPRVLQK 19 669 VRVPPSESQK 19 802 DVLPFIIPHR 195 LTLATEQPVK 18 30 ALLVIMSLGL 18 36 SLGLGLGLGL 18 160 PVILFSMDGF 18 173YLYTWDTLMP 18 215 GLYPESHGII 18 260 MYQGLKAATY 18 364 CVNIILLADH 18 426NLSCRKPDQH 18 447 RLHYAKNVRI 18 453 NVRIDKVHLF 18 697 ASNRTSDSQY 18 825ALWVEERFTA 18 12 PVKKNTLKKY 17 41 LGLGLRKLEK 17 46 RKLEKQGSCR 17 121QKKDCCADYK 17 233 LNKNFSLSSK 17 311 AERPRFYTMY 17 405 RIRAHNIPHD 17 458KVHLFVDQQW 17 469 AVRSKSNTNC 17 524 LLRIQPAPNN 17 544 KVPFYEPSHA 17 637AMRMPMWSSY 17 650 QLGDTSPLPP 17 670 RVPPSESQKC 17 692 FLYPPASNRT 17 713NLVPMYEEFR 17 742 GVNVVSGPIF 17 831 RFTAHIARVR 17 22 KIACIVLLAL 16 25CIVLLALLVI 16 67 ENCRCDVACK 16 113 CSCSDDCLQK 16 284 SIYMPYNGSV 16 295FEERISTLLK 16 341 ALQVVDHAFG 16 423 IVRNLSCRKP 16 455 RIDKVHLFVD 16 549EPSHAEEVSK 16 603 TVKVNLPFGR 16 614 RVLQKNVDHC 16 623 CLLYHREYVS 16 744NVVSGPIFDY 16 758 HFDAPDEITK 16 775 PIPTHYFVVL 16 783 VLTSCKNKSH 16 806FIIPHRPTNV 16 835 HIARVRDVEL 16 854 KVQPVSEILQ 16 861 ILQLKTYLPT 16 11QPVKKNTLKK 15 27 VLLALLVIMS 15 28 LLALLVIMSL 15 55 RKKCFDASFR 15 65GLENCRCDVA 15 131 SVCQGETSWL 15 157 DLPPVILFSM 15 161 VILFSMDGFR 15 162ILFSMDGFRA 15 223 IIDNNMYDVN 15 232 NLNKNFSLSS 15 285 IYMPYNGSVP 15 307DLPKAERPRF 15 331 GGPVSARVIK 15 343 QVVDHAFGML 15 367 IILLADHGMD 15 369LLADHGMDQT 15 516 EVYNLMCDLL 15 526 RIQPAPNNGT 15 607 NLPFGRPRVL 15 660TVPDCLRADV 15 668 DVRVPPSESQ 15 677 QKCSFYLADK 15 709 LITSNLVPMY 15 764EITKHLANTD 15 844 LLTGLDFYQD 15 863 QLKTYLPTFE 15 17 TLKKYKIACI 14 263GLKAATYFWP 14 292 SVPFEERIST 14 305 WLDLPKAERP 14 344 VVDHAFGMLM 14 348AFGMLMEGLK 14 428 SCRKPDQHFK 14 439 YLTPDLPKRL 14 467 WLAVRSKSNT 14 555EVSKFSVCGF 14 567 PLPTESLDCF 14 579 HLQNSTQLEQ 14 599 EITATVKVNL 14 619NVDHCLLYHR 14 656 PLPPTVPDCL 14 807 IIPHRPTNVE 14 827 WVEERFTAHI 14 847GLDFYQDKVQ 14 857 PVSEILQLKT 14 44 GLRKLEKQGS 13 72 DVACKDRGDC 13 91ESTRIWMCNK 13 139 WLEENCDTAQ 13 192 IHSKYMRAMY 13 226 NNMYDVNLNK 13 230DVNLNKNFSL 13 267 ATYFWPGSEV 13 304 KWLDLPKAER 13 330 AGGPVSARVI 13 333PVSARVIKAL 13 335 SARVIKALQV 13 446 KRLHYAKNVR 13 490 RSMEAIFLAH 13 508EVEPFENIEV 13 527 IQPAPNNGTH 13 535 THGSLNHLLK 13 560 SVCGFANPLP 13 585QLEQVNQMLN 13 627 HREYVSGFGK 13 647 TVPQLGDTSP 13 682 YLADKNITHG 13 726DYFHSVLLIK 13 732 LLIKHATERN 13 749 PIFDYNYDGH 13 779 HYFVVLTSCK 13 834AHIARVRDVE 13 V2-HLA-A3-10mers- 161P2F10BEach peptide is a portion of SEQ ID NO: 83; each start position isspecified, the length of the peptide is 10 amino acids and the endposition for each peptide is the start position plus 9. Pos 1234567890score 9 QRKDCCADYK 17 7 CLQRKDCCAD 14 2 SCSDDCLQRK 12 8 LQRKDCCADY 12 1CSCSDDCLQR 10 V3-HLA-A3-10mers- 161P2F10BEach peptide is a portion of SEQ ID NO: 86; each start position isspecified, the length of the peptide is 10 amino acids and the endposition for each peptide is the start position plus 9. Pos 1234567890score 4 NVESCPGGKP 14 3 TNVESCPGGK 13 10 GGKPEALWVE 8 9 PGGKPEALWV 7V4-HLA-A3-10mers- 161P2F10B Each peptide is a portion of SEQ IDNO: 89; each start position is specified, the length of the peptideis 10 amino acids and the end position for each peptide is thestart position plus 9. Pos 1234567890 score 1 KTYLPTFETP 12 2 TYLPTFETPI6

TABLE XXXVIII V1-HLA-A26-10mers- 161P2F10BEach peptide is a portion of SEQ ID NO: 81; each start position isspecified, the length of the peptide is 10 amino acids and the endposition for each peptide is the start position plus 9. Pos 1234567890score 555 EVSKFSVCGF 32 178 DTLMPNINKL 29 744 NVVSGPIFDY 29 516EVYNLMCDLL 28 841 DVELLTGLDF 28 230 DVNLNKNFSL 27 297 ERISTLLKWL 27 12PVKKNTLKKY 25 343 QVVDHAFGML 25 32 LVIMSLGLGL 24 154 EGFDLPPVIL 23 333PVSARVIKAL 23 388 DYFPRINFFY 23 599 EITATVKVNL 23 795 ENCPGWLDVL 23 802DVLPFIIPHR 23 160 PVILFSMDGF 22 453 NVRIDKVHLF 22 508 EVEPFENIEV 22 719EEFRKMWDYF 22 709 LITSNLVPMY 21 131 SVCQGETSWL 20 275 EVAINGSFPS 20 293VPFEERISTL 20 488 EFRSMEAIFL 20 853 DKVQPVSEIL 20 72 DVACKDRGDC 19 105ETRLEASLCS 19 376 DQTYCNKMEY 19 421 EEIVRNLSCR 19 571 ESLDCFCPHL 19 668DVRVPPSESQ 19 685 DKNITHGFLY 19 742 GVNVVSGPIF 19 792 HTPENCPGWL 19 22KIACIVLLAL 18 92 STRIWMCNKF 18 136 ETSWLEENCD 18 312 ERPRFYTMYF 18 422EIVRNLSCRK 18 494 AIFLAHGPSF 18 513 ENIEVYNLMC 18 587 EQVNQMLNLT 18 598EEITATVKVN 18 621 DHCLLYHREY 18 659 PTVPDCLRAD 18 700 RTSDSQYDAL 18 771NTDVPIPTHY 18 773 DVPIPTHYFV 18 830 ERFTAHIARV 18 386 MTDYFPRINF 17 457DKVHLFVDQQ 17 629 EYVSGFGKAM 17 653 DTSPLPPTVP 17 726 DYFHSVLLIK 17 760DAPDEITKHL 17 838 RVRDVELLTG 17 87 DTCVESTRIW 16 89 CVESTRIWMC 16 145DTAQQSQCPE 16 210 YTIVTGLYPE 16 296 EERISTLLKW 16 307 DLPKAERPRF 16 510EPFENIEVYN 16 534 GTHGSLNHLL 16 574 DCFCPHLQNS 16 602 ATVKVNLPFG 16 763DEITKHLANT 16 764 EITKHLANTD 16 277 AINGSFPSIY 15 280 GSFPSIYMPY 15 400EGPAPRIRAH 15 493 EAIFLAHGPS 15 554 EEVSKFSVCG 15 646 YTVPQLGDTS 15 739ERNGVNVVSG 15 820 EGKPEALWVE 15 858 VSEILQLKTY 15 V2-HLA-A26-10mers-161P2F10B Each peptide is a portion of SEQ IDNO: 83; each start position is specified, the length of the peptideis 10 amino acids and the end position for each peptide is thestart position plus 9. Pos 1234567890 score 8 LQRKDCCADY 10 5 DDCLQRKDCC8 6 DCLQRKDCCA 8 2 SCSDDCLQRK 6 V3-HLA-A26-10mers- 161P2F10BEach peptide is a portion of SEQ ID NO: 86; each start position isspecified, the length of the peptide is 10 amino acids and the endposition for each peptide is the start position plus 9. Pos 1234567890score 4 NVESCPGGKP 14 6 ESCPGGKPEA 11 7 SCPGGKPEAL 10 2 PTNVESCPGG 8 3TNVESCPGGK 8 V4-HLA-A26-10mers- 161P2F10BEach peptide is a portion of SEQ ID NO: 89; each start position isspecified, the length of the peptide is 10 amino acids and the endposition for each peptide is the start position plus 9. Pos 1234567890score 1 KTYLPTFETP 9

TABLE XXXIX V1-HLA-B0702- 10mers-161P2F10BEach peptide is a portion of SEQ ID NO: 81; each start position isspecified, the length of the peptide is 10 amino acids and the endposition for each peptide is the start position plus 9. Pos 1234567890score 431 KPDQHFKPYL 23 530 APNNGTHGSL 22 648 VPQLGDTSPL 22 577CPHLQNSTQL 21 797 CPGWLDVLPF 21 152 CPEGFDLPPV 20 293 VPFEERISTL 20 776IPTHYFVVLT 20 500 GPSFKEKTEV 19 761 APDEITKHLA 19 246 NPAWWHGQPM 18 658PPTVPDCLRA 18 672 PPSESQKCSF 18 715 VPMYEEFRKM 18 774 VPIPTHYFVV 18 332GPVSARVIKA 17 804 LPFIIPHRPT 17 22 KIACIVLLAL 15 34 IMSLGLGLGL 15 549EPSHAEEVSK 15 608 LPFGRPRVLQ 15 11 QPVKKNTLKK 14 30 ALLVIMSLGL 14 313RPRFYTMYFE 14 333 PVSARVIKAL 14 401 GPAPRIRAHN 14 403 APRIRAHNIP 14 437KPYLTPDLPK 14 441 TPDLPKRLHY 14 488 EFRSMEAIFL 14 599 EITATVKVNL 14 612RPRVLQKNVD 14 642 MWSSYTVPQL 14 700 RTSDSQYDAL 14 775 PIPTHYFVVL 14 836IARVRDVELL 14 19 KKYKIACIVL 13 20 KYKIACIVLL 13 36 SLGLGLGLGL 13 154EGFDLPPVIL 13 165 SMDGFRAEYL 13 224 IDNNMYDVNL 13 287 MPYNGSVPFE 13 299ISTLLKWLDL 13 323 EPDSSGHAGG 13 390 FPRINFFYMY 13 418 FNSEEIVRNL 13 435HFKPYLTPDL 13 452 KNVRIDKVHL 13 510 EPFENIEVYN 13 528 QPAPNNGTHG 13 566NPLPTESLDC 13 640 MPMWSSYTVP 13 655 SPLPPTVPDC 13 684 ADKNITHGFL 13 694YPPASNRTSD 13 705 QYDALITSNL 13 724 MWDYFHSVLL 13 795 ENCPGWLDVL 13 808IPHRPTNVES 13 818 CPEGKPEALW 13 822 KPEALWVEER 13 835 HIARVRDVEL 13 23IACIVLLALL 12 32 LVIMSLGLGL 12 39 LGLGLGLRKL 12 57 KCFDASFRGL 12 171AEYLYTWDTL 12 206 FPNHYTIVTG 12 207 PNHYTIVTGL 12 248 AWWHGQPMWL 12 282FPSIYMPYNG 12 308 LPKAERPRFY 12 347 HAFGMLMEGL 12 352 LMEGLKQRNL 12 534GTHGSLNHLL 12 564 FANPLPTESL 12 571 ESLDCFCPHL 12 586 LEQVNQMLNL 12 661VPDCLRADVR 12 674 SESQKCSFYL 12 695 PPASNRTSDS 12 723 KMWDYFHSVL 12 811RPTNVESCPE 12 839 VRDVELLTGL 12 855 VQPVSEILQL 12 9 TEQPVKKNTL 11 28LLALLVIMSL 11 76 KDRGDCCWDF 11 99 NKFRCGETRL 11 101 FRCGETRLEA 11 103CGETRLEASL 11 131 SVCQGETSWL 11 158 LPPVILFSMD 11 201 YPTKTFPNHY 11 253QPMWLTAMYQ 11 271 WPGSEVAING 11 294 PFEERISTLL 11 297 ERISTLLKWL 11 325DSSGHAGGPV 11 330 AGGPVSARVI 11 343 QVVDHAFGML 11 411 IPHDFFSFNS 11 439YLTPDLPKRL 11 444 LPKRLHYAKN 11 511 PFENIEVYNL 11 515 IEVYNLMCDL 11 516EVYNLMCDLL 11 545 VPFYEPSHAE 11 555 EVSKFSVCGF 11 559 FSVCGFANPL 11 568LPTESLDCFC 11 607 NLPFGRPRVL 11 615 VLQKNVDHCL 11 656 PLPPTVPDCL 11 688ITHGFLYPPA 11 708 ALITSNLVPM 11 760 DAPDEITKHL 11 767 KHLANTDVPI 11 817SCPEGKPEAL 11 859 SEILQLKTYL 11 V2-HLA-B0702- 10mers-161P2F10BEach peptide is a portion of SEQ ID NO: 83; each start position isspecified, the length of the peptide is 10 amino acids and the endposition for each peptide is the start position plus 9. Pos 1234567890score 6 DCLQRKDCCA 6 8 LQRKDCCADY 4 1 CSCSDDCLQR 2 2 SCSDDCLQRK 2 4SDDCLQRKDC 2 10 RKDCCADYKS 2 3 CSDDCLQRKD 1 7 CLQRKDCCAD 1 V3-HLA-B0702-10mers-161P2F10B Each peptide is a portion of SEQ IDNO: 86; each start position is specified, the length of the peptideis 10 amino acids and the end position for each peptide is thestart position plus 9. Pos 1234567890 score 8 CPGGKPEALW 14 7 SCPGGKPEAL13 1 RPTNVESCPG 12 9 PGGKPEALWV 10 6 ESCPGGKPEA 9 V4-HLA-B0702-10mers-161P2F10B Each peptide is a portion of SEQ IDNO: 89; each start position is specified, the length of the peptideis 10 amino acids and the end position for each peptide is thestart position plus 9. Pos 1234567890 score 2 TYLPTFETPI 9

Table XL-V1-HLA-B08-10mers-161P2F10B Pos 1234567890 score No ResultsFound. Table XL-V2-HLA-B08-10mers-161P2F10B Pos 1234567890 score NoResults Found. Table XL-V3-HLA-B08-10mers-161P2F10B Pos 123456789 scoreNo Results Found. Table XL-V4-HLA-B08-10mers-161P2F10B Pos 123456789score No Results Found.

Table XLI-V1-HLA-B1510-10mers-161P2F10B Pos 1234567890 score No ResultsFound. Table XLI-V2-HLA-B1510-10mers-161P2F10B Pos 1234567890 score NoResults Found. Table XLI-V3-HLA-B1510-10mers-161P2F10B Pos 123456789score No Results Found. Table XLI-V4-HLA-B1510-10mers-161P2F10B Pos123456789 score No Results Found.

Table XLII-V1-HLA-B2705-10mers-161P2F10B Pos 1234567890 score No ResultsFound. Table XLII-V2-HLA-B2705-10mers-161P2F10B Pos 1234567890 score NoResults Found. Table XLII-V3-HLA-B2705-10mers-161P2F10B Pos 123456789score No Results Found. Table XLII-V4-HLA-B2705-10mers-161P2F10B Pos123456789 score No Results Found.

Table XLIII-V1-HLA-B2709-10mers-161P2F10B Pos 1234567890 score NoResults Found. Table XLIII-V2-HLA-B2709-10mers-161P2F10B Pos 1234567890score No Results Found. Table XLIII-V3-HLA-B2709-10mers-161P2F10B Pos123456789 score No Results Found. TableXLIII-V4-HLA-B2709-10mers-161P2F10B Pos 123456789 score No ResultsFound.

TABLE XLIV V1-HLA-B4402-10mers- 161P2F10BEach peptide is a portion of SEQ ID NO: 81; each start position isspecified, the length of the peptide is 10 amino acids and the endposition for each peptide is the start position plus 9. Pos 1234567890score 311 AERPRFYTMY 26 9 TEQPVKKNTL 25 171 AEYLYTWDTL 25 296 EERISTLLKW25 509 VEPFENIEVY 25 719 EEFRKMWDYF 25 859 SEILQLKTYL 25 487 NEFRSMEAIF24 842 VELLTGLDFY 23 674 SESQKCSFYL 22 586 LEQVNQMLNL 21 823 PEALWVEERF21 153 PEGFDLPPVI 20 515 IEVYNLMCDL 20 598 EEITATVKVN 20 718 YEEFRKMWDY20 297 ERISTLLKWL 18 333 PVSARVIKAL 18 154 EGFDLPPVIL 17 322 EEPDSSGHAG17 330 AGGPVSARVI 17 409 HNIPHDFFSF 17 795 ENCPGWLDVL 17 20 KYKIACIVLL16 57 KCFDASFRGL 16 178 DTLMPNINKL 16 293 VPFEERISTL 16 421 EEIVRNLSCR16 439 YLTPDLPKRL 16 494 AIFLAHGPSF 16 855 VQPVSEILQL 16 22 KIACIVLLAL15 30 ALLVIMSLGL 15 39 LGLGLGLRKL 15 155 GFDLPPVILF 15 277 AINGSFPSIY 15280 GSFPSIYMPY 15 312 ERPRFYTMYF 15 340 KALQVVDHAF 15 361 LHNCVNIILL 15388 DYFPRINFFY 15 399 YEGPAPRIRA 15 430 RKPDQHFKPY 15 553 AEEVSKFSVC 15607 NLPFGRPRVL 15 656 PLPPTVPDCL 15 697 ASNRTSDSQY 15 760 DAPDEITKHL 15763 DEITKHLANT 15 772 TDVPIPTHYF 15 775 PIPTHYFVVL 15 817 SCPEGKPEAL 15829 EERFTAHIAR 15 1 MESTLTLATE 14 12 PVKKNTLKKY 14 28 LLALLVIMSL 14 32LVIMSLGLGL 14 74 ACKDRGDCCW 14 99 NKFRCGETRL 14 141 EENCDTAQQS 14 147AQQSQCPEGF 14 160 PVILFSMDGF 14 165 SMDGFRAEYL 14 197 MRAMYPTKTF 14 208NHYTIVTGLY 14 218 PESHGIIDNN 14 248 AWWHGQPMWL 14 269 YFWPGSEVAI 14 295FEERISTLLK 14 353 MEGLKQRNLH 14 387 TDYFPRINFF 14 435 HFKPYLTPDL 14 441TPDLPKRLHY 14 453 NVRIDKVHLF 14 488 EFRSMEAIFL 14 516 EVYNLMCDLL 14 530APNNGTHGSL 14 555 EVSKFSVCGF 14 564 FANPLPTESL 14 571 ESLDCFCPHL 14 684ADKNITHGFL 14 700 RTSDSQYDAL 14 744 NVVSGPIFDY 14 791 SHTPENCPGW 14 858VSEILQLKTY 14 19 KKYKIACIVL 13 34 IMSLGLGLGL 13 36 SLGLGLGLGL 13 50KQGSCRKKCF 13 66 LENCRCDVAC 13 85 FEDTCVESTR 13 92 STRIWMCNKF 13 104GETRLEASLC 13 140 LEENCDTAQQ 13 164 FSMDGFRAEY 13 228 MYDVNLNKNF 13 240SSKEQNNPAW 13 242 KEQNNPAWWH 13 260 MYQGLKAATY 13 276 VAINGSFPSI 13 321FEEPDSSGHA 13 347 HAFGMLMEGL 13 360 NLHNCVNIIL 13 381 NKMEYMTDYF 13 384EYMTDYFPRI 13 386 MTDYFPRINF 13 418 FNSEEIVRNL 13 452 KNVRIDKVHL 13 458KVHLFVDQQW 13 533 NGTHGSLNHL 13 534 GTHGSLNHLL 13 554 EEVSKFSVCG 13 567PLPTESLDCF 13 570 TESLDCFCPH 13 597 QEEITATVKV 13 599 EITATVKVNL 13 637AMRMPMWSSY 13 642 MWSSYTVPQL 13 683 LADKNITHGF 13 716 PMYEEFRKMW 13 723KMWDYFHSVL 13 724 MWDYFHSVLL 13 738 TERNGVNVVS 13 746 VSGPIFDYNY 13 771NTDVPIPTHY 13 815 VESCPEGKPE 13 828 VEERFTAHIA 13 862 LQLKTYLPTF 13 866TYLPTFETTI 13 23 IACIVLLALL 12 25 CIVLLALLVI 12 48 LEKQGSCRKK 12 76KDRGDCCWDF 12 87 DTCVESTRIW 12 90 VESTRIWMCN 12 131 SVCQGETSWL 12 168GFRAEYLYTW 12 192 IHSKYMRAMY 12 201 YPTKTFPNHY 12 220 SHGIIDNNMY 12 224IDNNMYDVNL 12 241 SKEQNNPAWW 12 252 GQPMWLTAMY 12 255 MWLTAMYQGL 12 261YQGLKAATYF 12 262 QGLKAATYFW 12 286 YMPYNGSVPF 12 299 ISTLLKWLDL 12 307DLPKAERPRF 12 308 LPKAERPRFY 12 343 QVVDHAFGML 12 352 LMEGLKQRNL 12 370LADHGMDQTY 12 402 PAPRIRAHNI 12 406 IRAHNIPHDF 12 420 SEEIVRNLSC 12 431KPDQHFKPYL 12 476 TNCGGGNHGY 12 492 MEAIFLAHGP 12 504 KEKTEVEPFE 12 511PFENIEVYNL 12 512 FENIEVYNLM 12 538 SLNHLLKVPF 12 539 LNHLLKVPFY 12 548YEPSHAEEVS 12 550 PSHAEEVSKF 12 559 FSVCGFANPL 12 577 CPHLQNSTQL 12 616LQKNVDHCLL 12 617 QKNVDHCLLY 12 621 DHCLLYHREY 12 625 LYHREYVSGF 12 628REYVSGFGKA 12 648 VPQLGDTSPL 12 679 CSFYLADKNI 12 705 QYDALITSNL 12 712SNLVPMYEEF 12 792 HTPENCPGWL 12 797 CPGWLDVLPF 12 818 CPEGKPEALW 12 819PEGKPEALWV 12 835 HIARVRDVEL 12 836 IARVRDVELL 12 839 VRDVELLTGL 12 841DVELLTGLDF 12 V2-HLA-B4402-10mers- 161P2F10BEach peptide is a portion of SEQ ID NO: 83; each start position isspecified, the length of the peptide is 10 amino acids and the endposition for each peptide is the start position plus 9. Pos 1234567890score 8 LQRKDCCADY 10 4 SDDCLQRKDC 5 2 SCSDDCLQRK 4 3 CSDDCLQRKD 4V3-HLA-B4402-10mers- 161P2F10B Each peptide is a portion of SEQ IDNO: 86; each start position is specified, the length of the peptideis 10 amino acids and the end position for each peptide is thestart position plus 9. Pos 1234567890 score 7 SCPGGKPEAL 16 5 VESCPGGKPE13 8 CPGGKPEALW 12 V4-HLA-B4402-10mers- 161P2F10BEach peptide is a portion of SEQ ID NO: 89; each start position isspecified, the length of the peptide is 10 amino acids and the endposition for each peptide is the start position plus 9. Pos 1234567890score 2 TYLPTFETPI 12

Table XLV-V1-HLA-B5101-10mers-161P2F10B Pos 1234567890 score No ResultsFound. Table XLV-V2-HLA-B5101-10mers-161P2F10B Pos 1234567890 score NoResults Found. Table XLV-V3-HLA-B5101-10mers-161P2F10B Pos 123456789score No Results Found. Table XLV-V4-HLA-B5101-10mers-161P2F10B Pos123456789 score No Results Found.

TABLE XLVI V1-DRB1-0101-15mers- 161P2F10BEach peptide is a portion of SEQ ID NO: 81;each start position is specified, the lengthof the peptide is 15 amino acids and the endposition for each peptide is the start position plus 14. Pos123456789012345 score 207 PNHYTIVTGLYPESH 36 181 MPNINKLKTCGIHSK 35 486NNEFRSMEAIFLAHG 35 839 VRDVELLTGLDFYQD 33 703 DSQYDALITSNLVPM 32 42GLGLRKLEKQGSCRK 31 740 RNGVNVVSGPIFDYN 31 28 LLALLVIMSLGLGLG 30 160PVILFSMDGFRAEYL 30 797 CPGWLDVLPFIIPHR 30 858 VSEILQLKTYLPTFE 30 801LDVLPFIIPHRPTNV 29 393 INFFYMYEGPAPRIR 28 421 EEIVRNLSCRKPDQH 28 627HREYVSGFGKAMRMP 28 30 ALLVIMSLGLGLGLG 27 32 LVIMSLGLGLGLGLR 27 34IMSLGLGLGLGLRKL 27 364 CVNIILLADHGMDQT 27 522 CDLLRIQPAPNNGTH 27 23IACIVLLALLVIMSL 26 258 TAMYQGLKAATYFWP 26 273 GSEVAINGSFPSIYM 26 283PSIYMPYNGSVPFEE 26 300 STLLKWLDLPKAERP 26 545 VPFYEPSHAEEVSKF 26 645SYTVPQLGDTSPLPP 26 778 THYFVVLTSCKNKSH 26 26 IVLLALLVIMSLGLG 25 230DVNLNKNFSLSSKEQ 25 266 AATYFWPGSEVAING 25 292 SVPFEERISTLLKWL 25 303LKWLDLPKAERPRFY 25 350 GMLMEGLKQRNLHNC 25 433 DQHFKPYLTPDLPKR 25 459VHLFVDQQWLAVRSK 25 492 MEAIFLAHGPSFKEK 25 509 VEPFENIEVYNLMCD 25 536HGSLNHLLKVPFYEP 25 561 VCGFANPLPTESLDC 25 22 KIACIVLLALLVIMS 24 25CIVLLALLVIMSLGL 24 163 LFSMDGFRAEYLYTW 24 174 LYTWDTLMPNINKLK 24 194SKYMRAMYPTKTFPN 24 246 NPAWWHGQPMWLTAM 24 313 RPRFYTMYFEEPDSS 24 316FYTMYFEEPDSSGHA 24 333 PVSARVIKALQVVDH 24 342 LQVVDHAFGMLMEGL 24 387TDYFPRINFFYMYEG 24 392 RINFFYMYEGPAPRI 24 395 FFYMYEGPAPRIRAH 24 412PHDFFSFNSEEIVRN 24 437 KPYLTPDLPKRLHYA 24 514 NIEVYNLMCDLLRIQ 24 518YNLMCDLLRIQPAPN 24 539 LNHLLKVPFYEPSHA 24 542 LLKVPFYEPSHAEEV 24 589VNQMLNLTQEEITAT 24 605 KVNLPFGRPRVLQKN 24 722 RKMWDYFHSVLLIKH 24 798PGWLDVLPFIIPHRP 24 45 LRKLEKQGSCRKKCF 23 84 DFEDTCVESTRIWMC 23 129YKSVCQGETSWLEEN 23 191 GIHSKYMRAMYPTKT 23 210 YTIVTGLYPESHGII 23 272PGSEVAINGSFPSIY 23 284 SIYMPYNGSVPFEER 23 328 GHAGGPVSARVIKAL 23 464DQQWLAVRSKSNTNC 23 562 CGFANPLPTESLDCF 23 637 AMRMPMWSSYTVPQL 23 644SSYTVPQLGDTSPLP 23 651 LGDTSPLPPTVPDCL 23 687 NITHGFLYPPASNRT 23 804LPFIIPHRPTNVESC 23 1 MESTLTLATEQPVKK 22 18 LKKYKIACIVLLALL 22 20KYKIACIVLLALLVI 22 145 DTAQQSQCPEGFDLP 22 152 CPEGFDLPPVILFSM 22 155GFDLPPVILFSMDGF 22 171 AEYLYTWDTLMPNIN 22 380 CNKMEYMTDYFPRIN 22 400EGPAPRIRAHNIPHD 22 434 QHFKPYLTPDLPKRL 22 575 CFCPHLQNSTQLEQV 22 648VPQLGDTSPLPPTVP 22 654 TSPLPPTVPDCLRAD 22 777 PTHYFVVLTSCKNKS 22 825ALWVEERFTAHIARV 22 833 TAHIARVRDVELLTG 22 60 DASFRGLENCRCDVA 21 142ENCDTAQQSQCPEGF 21 186 KLKTCGIHSKYMRAM 21 451 AKNVRIDKVHLFVDQ 21 490RSMEAIFLAHGPSFK 21 665 LRADVRVPPSESQKC 21 704 SQYDALITSNLVPMY 21 739ERNGVNVVSGPIFDY 21 770 ANTDVPIPTHYFVVL 21 98 CNKFRCGETRLEASL 20 253QPMWLTAMYQGLKAA 20 443 DLPKRLHYAKNVRID 20 521 MCDLLRIQPAPNNGT 20 553AEEVSKFSVCGFANP 20 623 CLLYHREYVSGFGKA 20 631 VSGFGKAMRMPMWSS 20 718YEEFRKMWDYFHSVL 20 729 HSVLLIKHATERNGV 20 750 IFDYNYDGHFDAPDE 20 829EERFTAHIARVRDVE 20 15 KNTLKKYKIACIVLL 19 37 LGLGLGLGLRKLEKQ 19 153PEGFDLPPVILFSMD 19 158 LPPVILFSMDGFRAE 19 170 RAEYLYTWDTLMPNI 19 226NNMYDVNLNKNFSLS 19 305 WLDLPKAERPRFYTM 19 340 KALQVVDHAFGMLME 19 346DHAFGMLMEGLKQRN 19 363 NCVNIILLADHGMDQ 19 413 HDFFSFNSEEIVRNL 19 500GPSFKEKTEVEPFEN 19 556 VSKFSVCGFANPLPT 19 603 TVKVNLPFGRPRVLQ 19 691GFLYPPASNRTSDSQ 19 762 PDEITKHLANTDVPI 19 771 NTDVPIPTHYFVVLT 19 817SCPEGKPEALWVEER 19 2 ESTLTLATEQPVKKN 18 12 PVKKNTLKKYKIACI 18 29LALLVIMSLGLGLGL 18 102 RCGETRLEASLCSCS 18 136 ETSWLEENCDTAQQS 18 157DLPPVILFSMDGFRA 18 183 NINKLKTCGIHSKYM 18 214 TGLYPESHGIIDNNM 18 228MYDVNLNKNFSLSSK 18 255 MWLTAMYQGLKAATY 18 259 AMYQGLKAATYFWPG 18 267ATYFWPGSEVAINGS 18 297 ERISTLLKWLDLPKA 18 302 LLKWLDLPKAERPRF 18 318TMYFEEPDSSGHAGG 18 322 EEPDSSGHAGGPVSA 18 362 HNCVNIILLADHGMD 18 382KMEYMTDYFPRINFF 18 515 IEVYNLMCDLLRIQP 18 595 LTQEEITATVKVNLP 18 613PRVLQKNVDHCLLYH 18 640 MPMWSSYTVPQLGDT 18 662 PDCLRADVRVPPSES 18 681FYLADKNITHGFLYP 18 689 THGFLYPPASNRTSD 18 748 GPIFDYNYDGHFDAP 18 763DEITKHLANTDVPIP 18 847 GLDFYQDKVQPVSEI 18 848 LDFYQDKVQPVSEIL 18 16NTLKKYKIACIVLLA 17 21 YKIACIVLLALLVIM 17 24 ACIVLLALLVIMSLG 17 31LLVIMSLGLGLGLGL 17 36 SLGLGLGLGLRKLEK 17 80 DCCWDFEDTCVESTR 17 103CGETRLEASLCSCSD 17 126 CADYKSVCQGETSWL 17 139 WLEENCDTAQQSQCP 17 147AQQSQCPEGFDLPPV 17 189 TCGIHSKYMRAMYPT 17 209 HYTIVTGLYPESHGI 17 212IVTGLYPESHGIIDN 17 227 NMYDVNLNKNFSLSS 17 236 NFSLSSKEQNNPAWW 17 261YQGLKAATYFWPGSE 17 265 KAATYFWPGSEVAIN 17 282 FPSIYMPYNGSVPFE 17 286YMPYNGSVPFEERIS 17 324 PDSSGHAGGPVSARV 17 331 GGPVSARVIKALQVV 17 336ARVIKALQVVDHAFG 17 341 ALQVVDHAFGMLMEG 17 347 HAFGMLMEGLKQRNL 17 372DHGMDQTYCNKMEYM 17 394 NFFYMYEGPAPRIRA 17 396 FYMYEGPAPRIRAHN 17 491SMEAIFLAHGPSFKE 17 493 EAIFLAHGPSFKEKT 17 506 KTEVEPFENIEVYNL 17 519NLMCDLLRIQPAPNN 17 527 IQPAPNNGTHGSLNH 17 540 NHLLKVPFYEPSHAE 17 557SKFSVCGFANPLPTE 17 597 QEEITATVKVNLPFG 17 601 TATVKVNLPFGRPRV 17 602ATVKVNLPFGRPRVL 17 629 EYVSGFGKAMRMPMW 17 677 QKCSFYLADKNITHG 17 685DKNITHGFLYPPASN 17 725 WDYFHSVLLIKHATE 17 726 DYFHSVLLIKHATER 17 734IKHATERNGVNVVSG 17 780 YFVVLTSCKNKSHTP 17 812 PTNVESCPEGKPEAL 17 823PEALWVEERFTAHIA 17 836 IARVRDVELLTGLDF 17 842 VELLTGLDFYQDKVQ 17V2-DRB1-0101-15mers- 161P2F10BEach peptide is a portion of SEQ ID NO: 84;each start position is specified, the lengthof the peptide is 15 amino acids and the endposition for each peptide is the start position plus 14. Pos123456789012345 score 2 EASLCSCSDDCLQRK 16 7 SCSDDCLQRKDCCAD 9 12CLQRKDCCADYKSVC 9 13 LQRKDCCADYKSVCQ 9 14 QRKDCCADYKSVCQG 9 9SDDCLQRKDCCADYK 8 10 DDCLQRKDCCADYKS 8 8 CSDDCLQRKDCCADY 7V3-HLA-DRB1-0101-15mers- 161P2F10BEach peptide is a portion of SEQ ID NO: 87;each start position is specified, the lengthof the peptide is 15 amino acids and theend position for each peptide is the start position plus 14. Pos123456789012345 score 7 PTNVESCPGGKPEAL 25 12 SCPGGKPEALWVEER 19 4PHRPTNVESCPGGKP 14 10 VESCPGGKPEALWVE 14 V4-HLA-DRB1-0101-15mers-161P2F10B Each peptide is a portion of SEQ ID NO: 90;each start position is specified, the lengthof the peptide is 15 amino acids and theend position for each peptide is the start position plus 14. Pos123456789012345 score 2 ILQLKTYLPTFETPI 16 1 EILQLKTYLPTFETP 10

TABLE XLVII V1-HLA-DRB1-0301-15mers- 161P2F10BEach peptide is a portion of SEQ ID NO: 81;each start position is specified, the lengthof the peptide is 15 amino acids and the endposition for each peptide is the start position plus 14. Pos123456789012345 score 437 KPYLTPDLPKRLHYA 31 451 AKNVRIDKVHLFVDQ 31 662PDCLRADVRVPPSES 28 458 KVHLFVDQQWLAVRS 27 597 QEEITATVKVNLPFG 27 228MYDVNLNKNFSLSSK 26 350 GMLMEGLKQRNLHNC 26 536 HGSLNHLLKVPFYEP 26 305WLDLPKAERPRFYTM 25 358 QRNLHNCVNIILLAD 24 30 ALLVIMSLGLGLGLG 22 366NIILLADHGMDQTYC 22 28 LLALLVIMSLGLGLG 21 32 LVIMSLGLGLGLGLR 21 34IMSLGLGLGLGLRKL 21 158 LPPVILFSMDGFRAE 21 161 VILFSMDGFRAEYLY 21 517VYNLMCDLLRIQPAP 21 707 DALITSNLVPMYEEF 21 742 GVNVVSGPIFDYNYD 21 10EQPVKKNTLKKYKIA 20 20 KYKIACIVLLALLVI 20 109 EASLCSCSDDCLQKK 20 118DCLQKKDCCADYKSV 20 236 NFSLSSKEQNNPAWW 20 284 SIYMPYNGSVPFEER 20 408AHNIPHDFFSFNSEE 20 467 WLAVRSKSNTNCGGG 20 615 VLQKNVDHCLLYHRE 20 635GKAMRMPMWSSYTVP 20 780 YFVVLTSCKNKSHTP 20 839 VRDVELLTGLDFYQD 20 847GLDFYQDKVQPVSEI 20 855 VQPVSEILQLKTYLP 20 2 ESTLTLATEQPVKKN 19 26IVLLALLVIMSLGLG 19 38 GLGLGLGLRKLEKQG 19 42 GLGLRKLEKQGSCRK 19 74ACKDRGDCCWDFEDT 19 129 YKSVCQGETSWLEEN 19 153 PEGFDLPPVILFSMD 19 257LTAMYQGLKAATYFW 19 331 GGPVSARVIKALQVV 19 335 SARVIKALQVVDHAF 19 341ALQVVDHAFGMLMEG 19 349 FGMLMEGLKQRNLHN 19 433 DQHFKPYLTPDLPKR 19 486NNEFRSMEAIFLAHG 19 492 MEAIFLAHGPSFKEK 19 514 NIEVYNLMCDLLRIQ 19 524LLRIQPAPNNGTHGS 19 553 AEEVSKFSVCGFANP 19 565 ANPLPTESLDCFCPH 19 654TSPLPPTVPDCLRAD 19 679 CSFYLADKNITHGFL 19 697 ASNRTSDSQYDALIT 19 740RNGVNVVSGPIFDYN 19 773 DVPIPTHYFVVLTSC 19 781 FVVLTSCKNKSHTPE 19 812PTNVESCPEGKPEAL 19 833 TAHIARVRDVELLTG 19 835 HIARVRDVELLTGLD 19 852QDKVQPVSEILQLKT 19 163 LFSMDGFRAEYLYTW 18 177 WDTLMPNINKLKTCG 18 178DTLMPNINKLKTCGI 18 224 IDNNMYDVNLNKNFS 18 254 PMWLTAMYQGLKAAT 18 267ATYFWPGSEVAINGS 18 275 EVAINGSFPSIYMPY 18 292 SVPFEERISTLLKWL 18 296EERISTLLKWLDLPK 18 445 PKRLHYAKNVRIDKV 18 465 QQWLAVRSKSNTNCG 18 506KTEVEPFENIEVYNL 18 509 VEPFENIEVYNLMCD 18 582 NSTQLEQVNQMLNLT 18 586LEQVNQMLNLTQEEI 18 589 VNQMLNLTQEEITAT 18 605 KVNLPFGRPRVLQKN 18 613PRVLQKNVDHCLLYH 18 621 DHCLLYHREYVSGFG 18 628 REYVSGFGKAMRMPM 18 658PPTVPDCLRADVRVP 18 711 TSNLVPMYEEFRKMW 18 712 SNLVPMYEEFRKMWD 18 721FRKMWDYFHSVLLIK 18 731 VLLIKHATERNGVNV 18 7 LATEQPVKKNTLKKY 17 92STRIWMCNKFRCGET 17 219 ESHGIIDNNMYDVNL 17 282 FPSIYMPYNGSVPFE 17 290NGSVPFEERISTLLK 17 439 YLTPDLPKRLHYAKN 17 457 DKVHLFVDQQWLAVR 17 500GPSFKEKTEVEPFEN 17 518 YNLMCDLLRIQPAPN 17 558 KFSVCGFANPLPTES 17 573LDCFCPHLQNSTQLE 17 577 CPHLQNSTQLEQVNQ 17 583 STQLEQVNQMLNLTQ 17 590NQMLNLTQEEITATV 17 714 LVPMYEEFRKMWDYF 17 746 VSGPIFDYNYDGHFD 17 823PEALWVEERFTAHIA 17 857 PVSEILQLKTYLPTF 17 60 DASFRGLENCRCDVA 16 70RCDVACKDRGDCCWD 16 78 RGDCCWDFEDTCVES 16 94 RIWMCNKFRCGETRL 16 101FRCGETRLEASLCSC 16 122 KKDCCADYKSVCQGE 16 226 NNMYDVNLNKNFSLS 16 234NKNFSLSSKEQNNPA 16 346 DHAFGMLMEGLKQRN 16 384 EYMTDYFPRINFFYM 16 450YAKNVRIDKVHLFVD 16 532 NNGTHGSLNHLLKVP 16 599 EITATVKVNLPFGRP 16 678KCSFYLADKNITHGF 16 748 GPIFDYNYDGHFDAP 16 24 ACIVLLALLVIMSLG 15 48LEKQGSCRKKCFDAS 15 56 KKCFDASFRGLENCR 15 97 MCNKFRCGETRLEAS 15 162ILFSMDGFRAEYLYT 15 164 FSMDGFRAEYLYTWD 15 190 CGIHSKYMRAMYPTK 15 218PESHGIIDNNMYDVN 15 374 GMDQTYCNKMEYMTD 15 386 MTDYFPRINFFYMYE 15 413HDFFSFNSEEIVRNL 15 474 SNTNCGGGNHGYNNE 15 478 CGGGNHGYNNEFRSM 15 485YNNEFRSMEAIFLAH 15 670 RVPPSESQKCSFYLA 15 728 FHSVLLIKHATERNG 15 803VLPFIIPHRPTNVES 15 821 GKPEALWVEERFTAH 15 V2-DRB1-0301-15mers- 161P2F10BEach peptide is a portion of SEQ ID NO: 84;each start position is specified, the lengthof the peptide is 15 amino acids and the endposition for each peptide is the start position plus 14. Pos123456789012345 score 2 EASLCSCSDDCLQRK 20 11 DCLQRKDCCADYKSV 20 15RKDCCADYKSVCQGE 16 4 SLCSCSDDCLQRKDC 12 5 LCSCSDDCLQRKDCC 11 10DDCLQRKDCCADYKS 11 V3-HLA-DRB1-0301-15mers- 161P2F10BEach peptide is a portion of SEQ ID NO: 87;each start position is specified, the lengthof the peptide is 15 amino acids and theend position for each peptide is the start position plus 14. Pos123456789012345 score 7 PTNVESCPGGKPEAL 13 10 VESCPGGKPEALWVE 10 11ESCPGGKPEALWVEE 9 9 NVESCPGGKPEALWV 8 1 FIIPHRPTNVESCPG 7 3IPHRPTNVESCPGGK 6 V4-HLA-DRB1-0301-15mers- 161P2F10BEach peptide is a portion of SEQ ID NO: 90;each start position is specified, the lengthof the peptide is 15 amino acids and theend position for each peptide is the start position plus 14. Pos123456789012345 score 2 ILQLKTYLPTFETPI 12 1 EILQLKTYLPTFETP 10

TABLE XLVIII V1-HLA-DRB1-0401-15mers- 161P2F10BEach peptide is a portion of SEQ ID NO: 81;each start position is specified, the lengthof the peptide is 15 amino acids and the endposition for each peptide is the start position plus 14. Pos123456789012345 score 126 CADYKSVCQGETSWL 28 412 PHDFFSFNSEEIVRN 28 464DQQWLAVRSKSNTNC 28 631 VSGFGKAMRMPMWSS 28 691 GFLYPPASNRTSDSQ 28 703DSQYDALITSNLVPM 28 722 RKMWDYFHSVLLIKH 28 750 IFDYNYDGHFDAPDE 28 777PTHYFVVLTSCKNKS 28 778 THYFVVLTSCKNKSH 28 28 LLALLVIMSLGLGLG 26 181MPNINKLKTCGIHSK 26 213 VTGLYPESHGIIDNN 26 290 NGSVPFEERISTLLK 26 350GMLMEGLKQRNLHNC 26 445 PKRLHYAKNVRIDKV 26 458 KVHLFVDQQWLAVRS 26 506KTEVEPFENIEVYNL 26 524 LLRIQPAPNNGTHGS 26 583 STQLEQVNQMLNLTQ 26 589VNQMLNLTQEEITAT 26 823 PEALWVEERFTAHIA 26 855 VQPVSEILQLKTYLP 26 18LKKYKIACIVLLALL 22 60 DASFRGLENCRCDVA 22 80 DCCWDFEDTCVESTR 22 136ETSWLEENCDTAQQS 22 172 EYLYTWDTLMPNINK 22 174 LYTWDTLMPNINKLK 22 193HSKYMRAMYPTKTFP 22 203 TKTFPNHYTIVTGLY 22 253 QPMWLTAMYQGLKAA 22 266AATYFWPGSEVAING 22 279 NGSFPSIYMPYNGSV 22 346 DHAFGMLMEGLKQRN 22 382KMEYMTDYFPRINFF 22 387 TDYFPRINFFYMYEG 22 415 FFSFNSEEIVRNLSC 22 433DQHFKPYLTPDLPKR 22 447 RLHYAKNVRIDKVHL 22 482 NHGYNNEFRSMEAIF 22 509VEPFENIEVYNLMCD 22 573 LDCFCPHLQNSTQLE 22 678 KCSFYLADKNITHGF 22 679CSFYLADKNITHGFL 22 797 CPGWLDVLPFIIPHR 22 803 VLPFIIPHRPTNVES 22 847GLDFYQDKVQPVSEI 22 2 ESTLTLATEQPVKKN 20 4 TLTLATEQPVKKNTL 20 20KYKIACIVLLALLVI 20 23 IACIVLLALLVIMSL 20 24 ACIVLLALLVIMSLG 20 25CIVLLALLVIMSLGL 20 26 IVLLALLVIMSLGLG 20 42 GLGLRKLEKQGSCRK 20 45LRKLEKQGSCRKKCF 20 129 YKSVCQGETSWLEEN 20 155 GFDLPPVILFSMDGF 20 163LFSMDGFRAEYLYTW 20 177 WDTLMPNINKLKTCG 20 178 DTLMPNINKLKTCGI 20 197MRAMYPTKTFPNHYT 20 225 DNNMYDVNLNKNFSL 20 228 MYDVNLNKNFSLSSK 20 230DVNLNKNFSLSSKEQ 20 254 PMWLTAMYQGLKAAT 20 273 GSEVAINGSFPSIYM 20 282FPSIYMPYNGSVPFE 20 300 STLLKWLDLPKAERP 20 305 WLDLPKAERPRFYTM 20 316FYTMYFEEPDSSGHA 20 335 SARVIKALQVVDHAF 20 339 IKALQVVDHAFGMLM 20 342LQVVDHAFGMLMEGL 20 358 QRNLHNCVNIILLAD 20 364 CVNIILLADHGMDQT 20 365VNIILLADHGMDQTY 20 366 NIILLADHGMDQTYC 20 408 AHNIPHDFFSFNSEE 20 420SEEIVRNLSCRKPDQ 20 437 KPYLTPDLPKRLHYA 20 514 NIEVYNLMCDLLRIQ 20 517VYNLMCDLLRIQPAP 20 518 YNLMCDLLRIQPAPN 20 521 MCDLLRIQPAPNNGT 20 558KFSVCGFANPLPTES 20 570 TESLDCFCPHLQNST 20 577 CPHLQNSTQLEQVNQ 20 586LEQVNQMLNLTQEEI 20 592 MLNLTQEEITATVKV 20 637 AMRMPMWSSYTVPQL 20 639RMPMWSSYTVPQLGD 20 658 PPTVPDCLRADVRVP 20 662 PDCLRADVRVPPSES 20 666RADVRVPPSESQKCS 20 690 HGFLYPPASNRTSDS 20 714 LVPMYEEFRKMWDYF 20 721FRKMWDYFHSVLLIK 20 728 FHSVLLIKHATERNG 20 730 SVLLIKHATERNGVN 20 771NTDVPIPTHYFVVLT 20 833 TAHIARVRDVELLTG 20 852 QDKVQPVSEILQLKT 20 858VSEILQLKTYLPTFE 20 84 DFEDTCVESTRIWMC 18 99 NKFRCGETRLEASLC 18 103CGETRLEASLCSCSD 18 135 GETSWLEENCDTAQQ 18 168 GFRAEYLYTWDTLMP 18 186KLKTCGIHSKYMRAM 18 200 MYPTKTFPNHYTIVT 18 224 IDNNMYDVNLNKNFS 18 233LNKNFSLSSKEQNNP 18 237 FSLSSKEQNNPAWWH 18 276 VAINGSFPSIYMPYN 18 293VPFEERISTLLKWLD 18 319 MYFEEPDSSGHAGGP 18 355 GLKQRNLHNCVNIIL 18 370LADHGMDQTYCNKME 18 397 YMYEGPAPRIRAHNI 18 409 HNIPHDFFSFNSEEI 18 417SFNSEEIVRNLSCRK 18 457 DKVHLFVDQQWLAVR 18 463 VDQQWLAVRSKSNTN 18 483HGYNNEFRSMEAIFL 18 491 SMEAIFLAHGPSFKE 18 533 NGTHGSLNHLLKVPF 18 549EPSHAEEVSKFSVCG 18 562 CGFANPLPTESLDCF 18 574 DCFCPHLQNSTQLEQ 18 580LQNSTQLEQVNQMLN 18 593 LNLTQEEITATVKVN 18 604 VKVNLPFGRPRVLQK 18 609PFGRPRVLQKNVDHC 18 614 RVLQKNVDHCLLYHR 18 669 VRVPPSESQKCSFYL 18 682YLADKNITHGFLYPP 18 697 ASNRTSDSQYDALIT 18 704 SQYDALITSNLVPMY 18 733LIKHATERNGVNVVS 18 739 ERNGVNVVSGPIFDY 18 758 HFDAPDEITKHLANT 18 763DEITKHLANTDVPIP 18 802 DVLPFIIPHRPTNVE 18 844 LLTGLDFYQDKVQPV 18 292SVPFEERISTLLKWL 17 848 LDFYQDKVQPVSEIL 17 56 KKCFDASFRGLENCR 16 82CWDFEDTCVESTRIW 16 98 CNKFRCGETRLEASL 16 161 VILFSMDGFRAEYLY 16 166MDGFRAEYLYTWDTL 16 170 RAEYLYTWDTLMPNI 16 207 PNHYTIVTGLYPESH 16 226NNMYDVNLNKNFSLS 16 234 NKNFSLSSKEQNNPA 16 246 NPAWWHGQPMWLTAM 16 247PAWWHGQPMWLTAMY 16 258 TAMYQGLKAATYFWP 16 267 ATYFWPGSEVAINGS 16 268TYFWPGSEVAINGSF 16 302 LLKWLDLPKAERPRF 16 317 YTMYFEEPDSSGHAG 16 318TMYFEEPDSSGHAGG 16 392 RINFFYMYEGPAPRI 16 394 NFFYMYEGPAPRIRA 16 413HDFFSFNSEEIVRNL 16 459 VHLFVDQQWLAVRSK 16 486 NNEFRSMEAIFLAHG 16 493EAIFLAHGPSFKEKT 16 544 KVPFYEPSHAEEVSK 16 545 VPFYEPSHAEEVSKF 16 561VCGFANPLPTESLDC 16 607 NLPFGRPRVLQKNVD 16 623 CLLYHREYVSGFGKA 16 627HREYVSGFGKAMRMP 16 640 MPMWSSYTVPQLGDT 16 643 WSSYTVPQLGDTSPL 16 715VPMYEEFRKMWDYFH 16 724 MWDYFHSVLLIKHAT 16 725 WDYFHSVLLIKHATE 16 756DGHFDAPDEITKHLA 16 824 EALWVEERFTAHIAR 16 441 TPDLPKRLHYAKNVR 15 825ALWVEERFTAHIARV 15 15 KNTLKKYKIACIVLL 14 30 ALLVIMSLGLGLGLG 14 31LLVIMSLGLGLGLGL 14 32 LVIMSLGLGLGLGLR 14 34 IMSLGLGLGLGLRKL 14 36SLGLGLGLGLRKLEK 14 38 GLGLGLGLRKLEKQG 14 63 FRGLENCRCDVACKD 14 105ETRLEASLCSCSDDC 14 109 EASLCSCSDDCLQKK 14 137 TSWLEENCDTAQQSQ 14 158LPPVILFSMDGFRAE 14 160 PVILFSMDGFRAEYL 14 171 AEYLYTWDTLMPNIN 14 184INKLKTCGIHSKYMR 14 194 SKYMRAMYPTKTFPN 14 209 HYTIVTGLYPESHGI 14 210YTIVTGLYPESHGII 14 220 SHGIIDNNMYDVNLN 14 221 HGIIDNNMYDVNLNK 14 257LTAMYQGLKAATYFW 14 261 YQGLKAATYFWPGSE 14 284 SIYMPYNGSVPFEER 14 296EERISTLLKWLDLPK 14 299 ISTLLKWLDLPKAER 14 336 ARVIKALQVVDHAFG 14 341ALQVVDHAFGMLMEG 14 348 AFGMLMEGLKQRNLH 14 349 FGMLMEGLKQRNLHN 14 362HNCVNIILLADHGMD 14 367 IILLADHGMDQTYCN 14 372 DHGMDQTYCNKMEYM 14 383MEYMTDYFPRINFFY 14 390 FPRINFFYMYEGPAP 14 395 FFYMYEGPAPRIRAH 14 421EEIVRNLSCRKPDQH 14 451 AKNVRIDKVHLFVDQ 14 453 NVRIDKVHLFVDQQW 14 456IDKVHLFVDQQWLAV 14 489 FRSMEAIFLAHGPSF 14 492 MEAIFLAHGPSFKEK 14 494AIFLAHGPSFKEKTE 14 522 CDLLRIQPAPNNGTH 14 536 HGSLNHLLKVPFYEP 14 539LNHLLKVPFYEPSHA 14 540 NHLLKVPFYEPSHAE 14 542 LLKVPFYEPSHAEEV 14 553AEEVSKFSVCGFANP 14 565 ANPLPTESLDCFCPH 14 590 NQMLNLTQEEITATV 14 597QEEITATVKVNLPFG 14 605 KVNLPFGRPRVLQKN 14 613 PRVLQKNVDHCLLYH 14 621DHCLLYHREYVSGFG 14 628 REYVSGFGKAMRMPM 14 645 SYTVPQLGDTSPLPP 14 654TSPLPPTVPDCLRAD 14 668 DVRVPPSESQKCSFY 14 706 YDALITSNLVPMYEE 14 712SNLVPMYEEFRKMWD 14 731 VLLIKHATERNGVNV 14 740 RNGVNVVSGPIFDYN 14 743VNVVSGPIFDYNYDG 14 773 DVPIPTHYFVVLTSC 14 779 HYFVVLTSCKNKSHT 14 780YFVVLTSCKNKSHTP 14 781 FVVLTSCKNKSHTPE 14 798 PGWLDVLPFIIPHRP 14 800WLDVLPFIIPHRPTN 14 801 LDVLPFIIPHRPTNV 14 804 LPFIIPHRPTNVESC 14 836IARVRDVELLTGLDF 14 839 VRDVELLTGLDFYQD 14 841 DVELLTGLDFYQDKV 14 842VELLTGLDFYQDKVQ 14 845 LTGLDFYQDKVQPVS 14 V2-DR1-0401-15mers- 161P2F10BEach peptide is a portion of SEQ ID NO: 84;each start position is specified, the lengthof the peptide is 15 amino acids and the endposition for each peptide is the start position plus 14. Pos123456789012345 score 2 EASLCSCSDDCLQRK 14 5 LCSCSDDCLQRKDCC 12 7SCSDDCLQRKDCCAD 12 14 QRKDCCADYKSVCQG 12 1 LEASLCSCSDDCLQR 6 3ASLCSCSDDCLQRKD 6 4 SLCSCSDDCLQRKDC 6 6 CSCSDDCLQRKDCCA 6 11DCLQRKDCCADYKSV 6 12 CLQRKDCCADYKSVC 6 13 LQRKDCCADYKSVCQ 6 15RKDCCADYKSVCQGE 6

TABLE XLIX V1-HLA-DRB1-1101-15mers- 161P2F10BEach peptide is a portion of SEQ ID NO: 81;each start position is specified, the lengthof the peptide is 15 amino acids and the endposition for each peptide is the start position plus 14. Pos123456789012345 score 339 IKALQVVDHAFGMLM 27 42 GLGLRKLEKQGSCRK 26 518YNLMCDLLRIQPAPN 26 207 PNHYTIVTGLYPESH 24 302 LLKWLDLPKAERPRF 24 750IFDYNYDGHFDAPDE 24 392 RINFFYMYEGPAPRI 23 417 SFNSEEIVRNLSCRK 23 313RPRFYTMYFEEPDSS 22 662 PDCLRADVRVPPSES 22 160 PVILFSMDGFRAEYL 21 178DTLMPNINKLKTCGI 21 296 EERISTLLKWLDLPK 21 759 FDAPDEITKHLANTD 21 780YFVVLTSCKNKSHTP 21 823 PEALWVEERFTAHIA 21 227 NMYDVNLNKNFSLSS 20 421EEIVRNLSCRKPDQH 20 447 RLHYAKNVRIDKVHL 20 491 SMEAIFLAHGPSFKE 20 536HGSLNHLLKVPFYEP 20 728 FHSVLLIKHATERNG 20 798 PGWLDVLPFIIPHRP 20 801LDVLPFIIPHRPTNV 20 29 LALLVIMSLGLGLGL 19 31 LLVIMSLGLGLGLGL 19 56KKCFDASFRGLENCR 19 300 STLLKWLDLPKAERP 19 678 KCSFYLADKNITHGF 19 848LDFYQDKVQPVSEIL 19 858 VSEILQLKTYLPTFE 19 25 CIVLLALLVIMSLGL 18 60DASFRGLENCRCDVA 18 234 NKNFSLSSKEQNNPA 18 266 AATYFWPGSEVAING 18 380CNKMEYMTDYFPRIN 18 482 NHGYNNEFRSMEAIF 18 489 FRSMEAIFLAHGPSF 18 539LNHLLKVPFYEPSHA 18 544 KVPFYEPSHAEEVSK 18 597 QEEITATVKVNLPFG 18 631VSGFGKAMRMPMWSS 18 645 SYTVPQLGDTSPLPP 18 715 VPMYEEFRKMWDYFH 18 777PTHYFVVLTSCKNKS 18 778 THYFVVLTSCKNKSH 18 803 VLPFIIPHRPTNVES 18 836IARVRDVELLTGLDF 18 68 NCRCDVACKDRGDCC 17 93 TRIWMCNKFRCGETR 17 174LYTWDTLMPNINKLK 17 283 PSIYMPYNGSVPFEE 17 317 YTMYFEEPDSSGHAG 17 346DHAFGMLMEGLKQRN 17 463 VDQQWLAVRSKSNTN 17 515 IEVYNLMCDLLRIQP 17 556VSKFSVCGFANPLPT 17 691 GFLYPPASNRTSDSQ 17 725 WDYFHSVLLIKHATE 17 797CPGWLDVLPFIIPHR 17 92 STRIWMCNKFRCGET 16 126 CADYKSVCQGETSWL 16 136ETSWLEENCDTAQQS 16 258 TAMYQGLKAATYFWP 16 279 NGSFPSIYMPYNGSV 16 305WLDLPKAERPRFYTM 16 349 FGMLMEGLKQRNLHN 16 387 TDYFPRINFFYMYEG 16 393INFFYMYEGPAPRIR 16 397 YMYEGPAPRIRAHNI 16 438 PYLTPDLPKRLHYAK 16 464DQQWLAVRSKSNTNC 16 486 NNEFRSMEAIFLAHG 16 500 GPSFKEKTEVEPFEN 16 509VEPFENIEVYNLMCD 16 561 VCGFANPLPTESLDC 16 610 FGRPRVLQKNVDHCL 16 703DSQYDALITSNLVPM 16 718 YEEFRKMWDYFHSVL 16 802 DVLPFIIPHRPTNVE 16 830ERFTAHIARVRDVEL 16 12 PVKKNTLKKYKIACI 15 38 GLGLGLGLRKLEKQG 15 213VTGLYPESHGIIDNN 15 254 PMWLTAMYQGLKAAT 15 332 GPVSARVIKALQVVD 15 401GPAPRIRAHNIPHDF 15 427 LSCRKPDQHFKPYLT 15 458 KVHLFVDQQWLAVRS 15 465QQWLAVRSKSNTNCG 15 533 NGTHGSLNHLLKVPF 15 614 RVLQKNVDHCLLYHR 15 619NVDHCLLYHREYVSG 15 721 FRKMWDYFHSVLLIK 15 846 TGLDFYQDKVQPVSE 15 7LATEQPVKKNTLKKY 14 20 KYKIACIVLLALLVI 14 26 IVLLALLVIMSLGLG 14 39LGLGLGLRKLEKQGS 14 70 RCDVACKDRGDCCWD 14 94 RIWMCNKFRCGETRL 14 99NKFRCGETRLEASLC 14 114 SCSDDCLQKKDCCAD 14 180 LMPNINKLKTCGIHS 14 196YMRAMYPTKTFPNHY 14 210 YTIVTGLYPESHGII 14 243 EQNNPAWWHGQPMWL 14 257LTAMYQGLKAATYFW 14 290 NGSVPFEERISTLLK 14 303 LKWLDLPKAERPRFY 14 321FEEPDSSGHAGGPVS 14 365 VNIILLADHGMDQTY 14 405 RIRAHNIPHDFFSFN 14 441TPDLPKRLHYAKNVR 14 450 YAKNVRIDKVHLFVD 14 550 PSHAEEVSKFSVCGF 14 602ATVKVNLPFGRPRVL 14 628 REYVSGFGKAMRMPM 14 658 PPTVPDCLRADVRVP 14 682YLADKNITHGFLYPP 14 685 DKNITHGFLYPPASN 14 714 LVPMYEEFRKMWDYF 14 724MWDYFHSVLLIKHAT 14 727 YFHSVLLIKHATERN 14 740 RNGVNVVSGPIFDYN 14 771NTDVPIPTHYFVVLT 14 814 NVESCPEGKPEALWV 14 829 EERFTAHIARVRDVE 14 842VELLTGLDFYQDKVQ 14 23 IACIVLLALLVIMSL 13 27 VLLALLVIMSLGLGL 13 28LLALLVIMSLGLGLG 13 33 VIMSLGLGLGLGLRK 13 63 FRGLENCRCDVACKD 13 194SKYMRAMYPTKTFPN 13 225 DNNMYDVNLNKNFSL 13 350 GMLMEGLKQRNLHNC 13 434QHFKPYLTPDLPKRL 13 453 NVRIDKVHLFVDQQW 13 514 NIEVYNLMCDLLRIQ 13 540NHLLKVPFYEPSHAE 13 558 KFSVCGFANPLPTES 13 583 STQLEQVNQMLNLTQ 13 598EEITATVKVNLPFGR 13 621 DHCLLYHREYVSGFG 13 643 WSSYTVPQLGDTSPL 13 651LGDTSPLPPTVPDCL 13 726 DYFHSVLLIKHATER 13 731 VLLIKHATERNGVNV 13 766TKHLANTDVPIPTHY 13 812 PTNVESCPEGKPEAL 13 852 QDKVQPVSEILQLKT 13 855VQPVSEILQLKTYLP 13 V2-DRB1-1101-15mers- 161P2F10BEach peptide is a portion of SEQ ID NO: 84;each start position is specified, the lengthof the peptide is 15 amino acids and the endposition for each peptide is the start position plus 14. Pos123456789012345 score 7 SCSDDCLQRKDCCAD 14 15 RKDCCADYKSVCQGE 9 8CSDDCLQRKDCCADY 8 13 LQRKDCCADYKSVCQ 7 2 EASLCSCSDDCLQRK 6 9SDDCLQRKDCCADYK 6 10 DDCLQRKDCCADYKS 6 V3-HLA-DRB1-1101-15mers-161P2F10B Each peptide is a portion of SEQ ID NO: 87;each start position is specified, the lengthof the peptide is 15 amino acids and theend position for each peptide is the start position plus 14. Pos123456789012345 score 4 PHRPTNVESCPGGKP 14 9 NVESCPGGKPEALWV 14 7PTNVESCPGGKPEAL 13 3 IPHRPTNVESCPGGK 8 5 HRPTNVESCPGGKPE 7 1FIIPHRPTNVESCPG 6 15 GGKPEALWVEERFTA 6 V4-HLA-DRB1-1101-15mers-161P2F10B Each peptide is a portion of SEQ ID NO: 90;each start position is specified, the lengthof the peptide is 15 amino acids and theend position for each peptide is the start position plus 14. Pos123456789012345 score 2 ILQLKTYLPTFETPI 6

TABLE XLVIII V3-HLA-DR1-0401-15mers- 161P2F10BEach peptide is a portion of SEQ ID NO: 87;each start position is specified, the lengthof the peptide is 15 amino acids and theend position for each peptide is the start position plus 14. Pos123456789012345 score 1 FIIPHRPTNVESCPG 12 4 PHRPTNVESCPGGKP 12 7PTNVESCPGGKPEAL 8 5 HRPTNVESCPGGKPE 6 6 RPTNVESCPGGKPEA 6 9NVESCPGGKPEALWV 6 10 VESCPGGKPEALWVE 6 12 SCPGGKPEALWVEER 6 13CPGGKPEALWVEERF 6 14 PGGKPEALWVEERFT 6 15 GGKPEALWVEERFTA 6V4-HLA-DR1-0401-15mers- 161P2F10BEach peptide is a portion of SEQ ID NO: 90;each start position is specified, the lengthof the peptide is 15 amino acids and theend position for each peptide is the start position plus 14. Pos123456789012345 score 2 ILQLKTYLPTFETPI 8

TABLE L Properties of 161P2F10B Bioinformatic Feature Program OutcomeORF (includes stop ORF finder codon) # of amino acids 875 Transmembraneregion TM Pred One TM, aa 23-41 HMMTop One TM, aa 23-45 Sosui One TM, aa23-45 TMHMM One TM, aa 23-45 Signal Peptide Signal P None pI pI/MW tool6.12 Molecular weight pI/MW tool 100.09 kDa Localization PSORT Plasmamembrane 74% Golgi 30% PSORT II Endoplasmic 30.4% Golgi 21.7% MotifsPfam Somatomedin B, Type I phosphodiesterase/ nucleotide pyrophosphatasePrints Cell Attachement RGD Blocks Somatomedin B, DNA/RNA non-specificendonuclease, Prosite Somatomedin B

TABLE LI Nucleotide sequence of transcript variant161P2F10B v.6 (SEQ ID NO: 91)atacagtttc tctttgccag actagactaa agaaggagca ctactttatt ctgataaaac 60aggtctatgc agctaccagg acaatggaat ctacgttgac tttagcaacg gaacaacctg 120ttaagaagaa cactcttaag aaatataaaa tagcttgcat tgttcttctt gctttgctgg 180tgatcatgtc acttggatta ggcctggggc ttggactcag gaaactggaa aagcaaggca 240gctgcaggaa gaagtgcttt gatgcatcat ttagaggact ggagaactgc cggtgtgatg 300tggcatgtaa agaccgaggt gattgctgct gggattttga agacacctgt gtggaatcaa 360ctcgaatatg gatgtgcaat aaatttcgtt gtggagagac cagattagag gccagccttt 420gctcttgttc agatgactgt ttgcagagga aagattgctg tgctgactat aagagtgttt 480gccaaggaga aacctcatgg ctggaagaaa actgtgacac agcccagcag tctcagtgcc 540cagaagggtt tgacctgcca ccagttatct tgttttctat ggatggattt agagctgaat 600atttatacac atgggatact ttaatgccaa atatcaataa actgaaaaca tgtggaattc 660attcaaaata catgagagct atgtatccta ccaaaacctt cccaaatcat tacaccattg 720tcacgggctt gtatccggag tcacatggca tcattgacaa taatatgtat gatgtaaatc 780tcaacaagaa tttttcactt tcttcaaagg aacaaaataa tccagcctgg tggcatgggc 840aaccaatgtg gctgacagca atgtatcaag gtttaaaagc cgctacctac ttttggcccg 900gatcagaagt ggctataaat ggctcctttc cttccatata catgccttac aacggaagtg 960tcccatttga agagaggatt tctacactgt taaaatggct ggacctgccc aaagctgaga 1020gacccaggtt ttataccatg ttttttgaag aacctgattc ctctggacat gcaggtggac 1080cagtcagtgc cagagtaatt aaagccttac aggtagtaga tcatgctttt gggatgttga 1140tggaaggcct gaagcagcgg aatttgcaca actgtgtcaa tatcatcctt ctggctgacc 1200atggaatgga ccagacttat tgtaacaaga tggaatacat gactgattat tttcccagaa 1260taaacttctt ctacatgtac gaagggcctg ccccccgcgt ccgagctcat aatatacctc 1320atgacttttt tagttttaat tctgaggaaa ttgttagaaa cctcagttgc cgaaaacctg 1380atcagcattt caagccctat ttgactcctg atttgccaaa gcgactgcac tatgccaaga 1440acgtcagaat cgacaaagtt catctctttg tggatcaaca gtggctggct gttaggagta 1500aatcaaatac aaattgtgga ggaggcaacc atggttataa caatgagttt aggagcatgg 1560aggctatctt tctggcacat ggacccagtt ttaaagagaa gactgaagtt gaaccatttg 1620aaaatattga agtctataac ctaatgtgtg atcttctacg cattcaacca gcaccaaaca 1680atggaaccca tggtagttta aaccatcttc tgaaggtgcc tttttatgag ccatcccatg 1740cagaggaggt gtcaaagttt tctgtttgtg gctttgctaa tccattgccc acagagtctc 1800ttgactgttt ctgccctcac ctacaaaata gtactcagct ggaacaagtg aatcagatgc 1860taaatctcac ccaagaagaa ataacagcaa cagtgaaagt aaatttgcca tttgggaggc 1920ctagggtact gcagaagaac gtggaccact gtctccttta ccacagggaa tatgtcagtg 1980gatttggaaa agctatgagg atgcccatgt ggagttcata cacagtcccc cagttgggag 2040acacatcgcc tctgcctccc actgtcccag actgtctgcg ggctgatgtc agggttcctc 2100cttctgagag ccaaaaatgt tccttctatt tagcagacaa gaatatcacc cacggcttcc 2160tctatcctcc tgccagcaat agaacatcag atagccaata tgatgcttta attactagca 2220atttggtacc tatgtatgaa gaattcagaa aaatgtggga ctacttccac agtgttcttc 2280ttataaaaca tgccacagaa agaaatggag taaatgtggt tagtggacca atatttgatt 2340ataattatga tggccatttt gatgctccag atgaaattac caaacattta gccaacactg 2400atgttcccat cccaacacac tactttgtgg tgctgaccag ttgtaaaaac aagagccaca 2460caccggaaaa ctgccctggg tggctggatg tcctaccctt tatcatccct caccgaccta 2520ccaacgtgga gagctgtcct gaaggtaaac cagaagctct ttgggttgaa gaaagattta 2580cagctcacat tgcccgggtc cgtgatgtag aacttctcac tgggcttgac ttctatcagg 2640ataaagtgca gcctgtctct gaaattttgc aactaaagac atatttacca acatttgaaa 2700ccactattta acttaataat gtctacttaa tatataattt actgtataaa gtaattttgg 2760caaaatataa gtgatttttt tctggagaat tgtaaaataa agttttctat ttttccttaa 2820gtcccctaaa agccataatt tttattattc ctttttctct tttttcaatt ctatgaatat 2880gtattatttt aaagttatat ttttcacaca gagatgatgc tatattacac cttccctttt 2940ttgttggttt cttaaactct aatctcatga cagattatac cttccttatt acttgtttta 3000tcttactcag aatctttgaa tatatttttc tgcccagaat tatctaaaca aaagggagaa 3060caaaagaagt atgtctcact tgggaactga atcaactcta aatcagtttt gtcacaaaac 3120tttttgtatt tgactggcaa tgctgattaa aattaaaaat gcaca 3165

TABLE LII Nucleotide sequence alignment of 161P2F10B v.1 (SEQ ID NO: 92)and 161P2F10B v.6 (SEQ ID NO: 93) Score = 5301 bits (2757), Expect =0.0Identities = 2774/2780 (99%), Gaps = 1/2780 (0%) Strand = Plus/Plus

TABLE LIII Peptide sequences of protein coded by 161P2F2F10B v.6(SEQ ID NO: 94)MESTLTLATE QPVKKNTLKK YKIACIVLLA LLVIMSLGLG LGLGLRKLEK QGSCRKKCFD 60ASFRGLENCR CDVACKDRGD CCWDFEDTCV ESTRIWMCNK FRCGETRLEA SLCSCSDDCL 120QKKDCCADYK SVCQGETSWL EENCDTAQQS QCPEGFDLPP VILFSMDGFR AEYLYTWDTL 180MPNINKLKTC GIHSKYMRAM YPTKTFPNHY TIVTGLYPES HGIIDNNMYD VNLNKNFSLS 240SKEQNNPAWW HGQPMWLTAM YQGLKAATYF WPGSEVAING SFPSIYMPYN GSVPFEERIS 300TLLKWLDLPK AERPREYTMY FEEPDSSGHA GGPVSARVIK ALQVVDHAFG MLMEGLKQRN 360LHNCVNIILL ADHGMDQTYC NKMEYMTDYF PRINFFYMYE GPAPRIRAHN IPHDFFSFNS 420EEIVRNLSCR KPDQHFKPYL TPDLPKRLHY AKNVRIDKVH LFVDQQWLAV RSKSNTNCGG 480GNHGYNNEFR SMEAIFLAHG PSFKEKTEVE PFENIEVYNL MCDLLRIQPA PNNGTHGSLN 540HLLKVPFYEP SHAEEVSKFS VCGFANPLPT ESLDCFCPHL QNSTQLEQVN QMLNLTQEEI 600TATVKVNLPF GRPRVLQKNV DHCLLYHREY VSGFGKAMRM PMWSSYTVPQ LGDTSPLPPT 660VPDCLRADVR VPPSESQKCS FYLADKNITH GFLYPPASNR TSDSQYDALI TSNLVPMYEE 720FRKMWDYFHS VLLIKHATER NGVNVVSGPI FDYNYDGHFD APDEITKHLA NTDVPIPTHY 780FVVLTSCKNK SHTPENCPGW LDVLPFIIPH RPTNVESCPE GKPEALWVEE RFTAHIARVR 840DVELLTGLDF YQDKVQPVSE ILQLKTYLPT FEITI 875

TABLE LIVAmino acid sequence alignment of 161P2F10Bv.1 v.1 (SEQ ID NO: 95) and161P2F10B v.6 (SEQ ID NO: 96) Score = 1855 bits (4804), Expect =0.0 Identities = 875/875 (100%), Positives = 875/875 (100%)161P2F10Bv.1: 1MESTLTLATEQPVKKNTLKKYKIACIVLLALLVIMSLGLGLGLGLRKLEKQGSCRKKCFD 60MESTLTLATEQPVKKNTLKKYKIACIVLLALLVIMSLGLGLGLGLRKLEKQGSCRKKCFD161P2F10Bv.6: 1MESTLTLATEQPVKKNTLKKYKIACIVLLALLVIMSLGLGLGLGLRKLEKQGSCRKKCFD 60161P2F10Bv.1: 61ASFRGLENCRCDVACKDRGDCCWDFEDTCVESTRIWMCNKFRCGETRLEASLCSCSDDCL 120ASFRGLENCRCDVACKDRGDCCWDFEDTCVESTRIWMCNKFRCGETRLEASLCSCSDDCL161P2F10Bv.6: 61ASFRGLENCRCDVACKDRGDCCWDFEDTCVESTRIWMCNKFRCGETRLEASLCSCSDDCL 120161P2F10Bv.1: 121QKKDCCADYKSVCQGETSWLEENCDTAQQSQCPEGFDLPPVILFSMDGFRAEYLYTWDTL 180QKKDCCADYKSVCQGETSWLEENCDTAQQSQCPEGFDLPPVILFSMDGFRAEYLYTWDTL161P2F10Bv.6: 121QKKDCCADYKSVCQGETSWLEENCDTAQQSQCPEGFDLPPVILFSMDGFRAEYLYTWDTL 180161P2F10Bv.1: 181MPNINKLKTCGIHSKYMRAMYPTKTFPNHYTIVTGLYPESHGIIDNNMYDVNLNKNFSLS 240MPNINKLKTCGIHSKYMRAMYPTKTFPNHYTIVTGLYPESHGIIDNNMYDVNLNKNFSLS161P2F10Bv.6: 181MPNINKLKTCGIHSKYMRAMYPTKTFPNHYTIVTGLYPESHGIIDNNMYDVNLNKNFSLS 240161P2F10Bv.1: 241SKEQNNPAWWHGQPMWLTAMYQGLKAATYFWPGSEVAINGSFPSIYMPYNGSVPFEERIS 300SKEQNNPAWWHGQPMWLTAMYQGLKAATYFWPGSEVAINGSFPSIYMPYNGSVPFEERIS161P2F10Bv.6: 241SKEQNNPAWWHGQPMWLTAMYQGLKAATYFWPGSEVAINGSFPSIYMPYNGSVPFEERIS 300161P2F10Bv.1: 301TLLKWLDLPKAERPRFYTMYFEEPDSSGHAGGPVSARVIKALQVVDHAFGMLMEGLKQRN 360TLLKWLDLPKAERPRFYTMYFEEPDSSGHAGGPVSARVIKALQVVDHAFGMLMEGLKQRN161P2F10Bv.6: 301TLLKWLDLPKAERPRFYTMYFEEPDSSGHAGGPVSARVIKALQVVDHAFGMLMEGLKQRN 360161P2F10Bv.1: 361LHNCVNIILLADHGMDQTYCNKMEYMTDYFPRINFFYMYEGPAPRIRAHNIPHDFFSFNS 420LHNCVNIILLADHGMDQTYCNKMEYMTDYFPRINFFYMYEGPAPRIRAHNIPHDFFSFNS161P2F10Bv.6: 361LHNCVNIILLADHGMDQTYCNKMEYMTDYFPRINFFYMYEGPAPRIRAHNIPHDFFSFNS 420161P2F10Bv.1: 421EETVRNLSCRKPDQHFKPYLTPDLPKRLHYAKNVRIDKVHLFVDQQWLAVRSKSNTNCGG 480EETVRNLSCRKPDQHFKPYLTPDLPKRLHYAKNVRIDKVHLFVDQQWLAVRSKSNTNCGG161P2F10Bv.6: 421EETVRNLSCRKPDQHFKPYLTPDLPKRLHYAKNVRIDKVHLFVDQQWLAVRSKSNTNCGG 480161P2F10Bv.1: 481GNHGYNNEFRSMEAIFLAHGPSFKEKTEVEPFENIEVYNLMCDLLRIQPAPNNGTHGSLN 540GNHGYNNEFRSMEAIFLAHGPSFKEKTEVEPFENIEVYNLMCDLLRIQPAPNNGTHGSLN161P2F10Bv.6: 481GNHGYNNEFRSMEAIFLAHGPSFKEKTEVEPFENIEVYNLMCDLLRIQPAPNNGTHGSLN 540161P2F10Bv.1: 541HLLKVPFYEPSHAEEVSKFSVCGFANPLPTESLDCFCPHLQNSTQLEQVNQMLNLTQEEI 600HLLKVPFYEPSHAEEVSKFSVCGFANPLPTESLDCFCPHLQNSTQLEQVNQMLNLTQEEI161P2F10Bv.6: 541HLLKVPFYEPSHAEEVSKFSVCGFANPLPTESLDCFCPHLQNSTQLEQVNQMLNLTQEEI 600161P2F10Bv.1: 601TATVKVNLPFGRPRVLQKNVDHCLLYHREYVSGFGKAMRMPMWSSYTVPQLGDTSPLPPT 660TATVKVNLPFGRPRVLQKNVDHCLLYHREYVSGFGKAMRMPMWSSYTVPQLGDTSPLPPT161P2F10Bv.6: 601TATVKVNLPFGRPRVLQKNVDHCLLYHREYVSGFGKAMRMPMWSSYTVPQLGDTSPLPPT 660161P2F10Bv.1: 661VPDCLFADVRVPPSESQKCSFYLADKNITHGFLYPPASNRTSDSQYDALITSNLVPMYEE 720VPDCLRADVRVPPSESQKCSFYLADKNITHGFLYPPASNRTSDSQYDALITSNLVPMYEE161P2F10Bv.6: 661VPDCLFADVRVPPSESQKCSFYLADKNITHGFLYPPASNRTSDSQYDALITSNLVPMYEE 720161P2F10Bv.1: 721FRKMWDYFHSVLLIKHATERNGVNVVSGPIFDYNYDGHFDAPDEITKHLANTDVPIPTHY 780FRKMWDYFHSVLLIKHATERNGVNVVSGPIFDYNYDGHFDAPDEITKHLANTDVPIPTHY161P2F10Bv.6: 721FRKMWDYFHSVLLIKHATERNGVNVVSGPIFDYNYDGHFDAPDEITKHLANTDVPIPTHY 780161P2F10Bv.1: 781FVVLTSCKNKSHTPENCPGWLDVLPFIIPHRPTNVESCPEGKPEALWVEERFTAHIARVR 840FVVLTSCKNKSHTPENCPGWLDVLPFIIPHRPTNVESCPEGKPEALWVEERFTAHIARVR161P2F10Bv.6: 781FVVLTSCKNKSHTPENCPGWLDVLPFIIPHRPTNVESCPEGKPEALWVEERFTAHIARVR 840161P2F10Bv.1: 841 DVELLTGLDFYQDKVQPVSEILQLKTYLPTFETTI 875DVELLTGLDFYQDKVQPVSEILQLKTYLPTFETTI 161P2F10Bv.6: 841DVELLTGLDFYQDKVQPVSEILQLKTYLPTFETTI 875

TABLE LV Nucleotide sequence of transcript variant 1612P2F10B v.7(SEQ ID NO: 97)ctactttatt ctgataaaac aggtctatgc agctaccagg acaatggaat ctacgttgac 60tttagcaacg gaacaacctg ttaagaagaa cactcttaag aaatataaaa tagcttgcat 120tacagggtct ctctcctttg ggatctcacc tcaccacaac ctctgtttcc caggctcaag 180tgatcctcct gcctcagcct cctgagtagc ttggaccaca ggcacatgcc acaaggctca 240gctaagtttt tgttcttctt gctttgctgg tgatcatgtc acttggatta ggcctggggc 300ttggactcag gaaactggaa aagcaaggca gctgcaggaa gaagtgcttt gatgcatcat 360ttagaggact ggagaactgc cggtgtgatg tggcatgtaa agaccgaggt gattgctgct 420gggattttga agacacctgt gtggaatcaa ctcgaatatg gatgtgcaat aaatttcgtt 480gtggagagac cagattagag gccagccttt gctcttgttc agatgactgt ttgcagaaga 540aagattgctg tgctgactat aagagtgttt gccaaggaga aacctcatgg ctggaagaaa 600actgtgacac agcccagcag tctcagtgcc cagaagggtt tgacctgcca ccagttatct 660tgttttctat ggatggattt agagctgaat atttatacac atgggatact ttaatgccaa 720atatcaataa actgaaaaca tgtggaattc attcaaaata catgagagct atgtatccta 780ccaaaacctt cccaaatcat tacaccattg tcacgggctt gtatccagag tcacatggca 840tcattgacaa taatatgtat gatgtaaatc tcaacaagaa tttttcactt tcttcaaagg 900aacaaaataa tccagcctgg tggcatgggc aaccaatgtg gctgacagca atgtatcaag 960gtttaaaagc cgctacctac ttttggcccg gatcagaagt ggctataaat ggctcctttc 1020cttccatata catgccttac aacggaagtg tcccatttga agagaggatt tctacactgt 1080taaaatggct ggacctgccc aaagctgaaa gacccaggtt ttataccatg tattttgaag 1140aacctgattc ctctggacat gcaggtggac cagtcagtgc cagagtaatt aaagccttac 1200aggtagtaga tcatgctttt gggatgttga tggaaggcct gaagcagcgg aatttgcaca 1260actgtgtcaa tatcatcctt ctggctgacc atggaatgga ccagacttat tgtaacaaga 1320tggaatacat gactgattat tttcccagaa taaacttctt ctacatgtac gaagggcctg 1380ccccccgcat ccgagctcat aatatacctc atgacttttt tagttttaat tctgaggaaa 1440ttgttagaaa cctcagttgc cgaaaacctg atcagcattt caagccctat ttgactcctg 1500atttgccaaa gcgactgcac tatgccaaga acgtcagaat cgacaaagtt catctctttg 1560tggatcaaca gtggctggct gttaggagta aatcaaatac aaattgtgga ggaggcaacc 1620atggttataa caatgagttt aggagcatgg aggctatctt tctggcacat ggacccagtt 1680ttaaagagaa gactgaagtt gaaccatttg aaaatattga agtctataac ctaatgtgtg 1740atcttctacg cattcaacca gcaccaaaca atggaaccca tggtagttta aaccatcttc 1800tgaaggtgcc tttttatgag ccatcccatg cagaggaggt gtcaaagttt tctgtttgtg 1860gctttgctaa tccattgccc acagagtctc ttgactgttt ctgccctcac ctacaaaata 1920gtactcagct ggaacaagtg aatcagatgc taaatctcac ccaagaagaa ataacagcaa 1980cagtgaaagt aaatttgcca tttgggaggc ctagggtact gcagaagaac gtggaccact 2040gtctccttta ccacagggaa tatgtcagtg gatttggaaa agctatgagg atgcccatgt 2100ggagttcata cacagtcccc cagttgggag acacatcgcc tctgcctccc actgtcccag 2160actgtctgcg ggctgatgtc agggttcctc cttctgagag ccaaaaatgt tccttctatt 2220tagcagacaa gaatatcacc cacggcttcc tctatcctcc tgccagcaat agaacatcag 2280atagccaata tgatgcttta attactagca atttggtacc tatgtatgaa gaattcagaa 2340aaatgtggga ctacttccac agtgttcttc ttataaaaca tgccacagaa agaaatggag 2400taaatgtggt tagtggacca atatttgatt ataattatga tggccatttt gatgctccag 2460atgaaattac caaacattta gccaacactg atgttcccat cccaacacac tactttgtgg 2520tgctgaccag ttgtaaaaac aagagccaca caccggaaaa ctgccctggg tggctggatg 2580tcctaccctt tatcatccct caccgaccta ccaacgtgga gagctgtcct gaaggtaaac 2640cagaagctct ttgggttgaa gaaagattta cagctcacat tgcccgggtc cgtgatgtag 2700aacttctcac tgggcttgac ttctatcagg ataaagtgca gcctgtctct gaaattttgc 2760aactaaagac atatttacca acatttgaaa ccactattta acttaataat gtctacttaa 2820tatataattt actgtataaa gtaattttgg caaaatataa gtgatttttt ctggagaatt 2880gtaaaataaa gttttctatt tttccttaaa aaaaaaaccg gaattccggg cttgggaggc 2940tgaggcagga gactcgcttg aacccgggag gcagaggttg cagtgagcca agattgcgcc 3000attgcactcc agagcctggg tgacagagca agactacatc tcaaaaaata aataaataaa 3060ataaaagtaa caataaaaat aaaaagaaca gcagagagaa tgagcaagga gaaatgtcac 3120aaactattgc aaaatactgt tacactgggt tggctctcca agaagatact ggaatctctt 3180cagccatttg cttttcagaa gtagaaacca gcaaaccacc tctaagcgga gaacatacga 3240ttctttatta agtagctctg gggaaggaaa gaataaaagt tgatagctcc ctgattggga 3300aaaaatgcac aattaataaa gaatgaagat gaaagaaagc atgcttatgt tgtaacacaa 3360aaaaaattca caaacgttgg tggaaggaaa acagtataga aaacattact ttaactaaaa 3420gctggaaaaa ttttcagttg ggatgcgact gacaaaaaga acgggatttc caggcataaa 3480gttggcgtga gctacagagg gcaccatgtg gctcagtgga agacccttca agattcaaag 3540ttccatttga cagagcaaag gcacttcgca aggagaaggg tttaaattat gggtccaaaa 3600gccaagtggt aaagcgagca atttgcagca taactgcttc tcctagacag ggctgagtgg 3660gcaaaatacg acagtacaca cagtgactat tagccactgc cagaaacagg ctgaacagcc 3720ctgggagaca agggaaggca ggtggtggga gttgttcatg gagagaaagg agagttttag 3780aaccagcaca tccactggag atgctgggcc accagacccc tcccagtcaa taaagtctgg 3840tgcctcattt gatctcagcc tcatcatgac cctggagaga ccctgatacc atctgccagt 3900ccccgacagc ttaggcactc cttgccatca acctgacccc ccgagtggtt ctccaggctc 3960cctgccccac ccattcaggc cggaattc 3988

TABLE LVI Nucleotide sequence alignment of 161P2F10B v.1 (SEQ ID NO: 98)and 161P2F10B v.7 (SEQ ID NO: 99) Score = 233 bits (121), Expect =2e-57Identities = 121/121 (100%) Strand = Plus/ Plus

Score = 7189 bits (3739), Expect = 0.0Identities = 3739/3739 (100%)Strand = Plus/ Plus

TABLE LVIIPeptide sequences of protein coded by 161P2F10B v.7 (SEQ ID NO: 100)MSLGLGLGLG LRKLEKQGSC RKKCFDASFR GLENCRCDVA CKDRGDCCWD FEDTCVESTR 60IWMCNKFRCG ETRLEASLCS CSDDCLQRKD CCADYKSVCQ GETSWLEENC DTAQQSQCPE 120GFDLPPVILF SMDGFRAEYL YTWDTLMPNI NKLKTCGIHS KYMRAMYPTK TFPNHYTIVT 180GLYPESHGII DNNMYDVNLN KNFSLSSKEQ NNPAWWHGQP MWLTAMYQGL KAATYFWPGS 240EVAINGSFPS IYMPYNGSVP FEERISTLLK WLDLPKAERP RFYTMFFEEP DSSGHAGGPV 300SARVIKALQV VDHAFGMLME GLKQRNLHNC VNIILLADHG MDQTYCNKME YMTDYFPRIN 360FFYMYEGPAP RVRAHNIPHD FFSFNSEEIV RNLSCRKPDQ HFKPYLTPDL PKRLHYAKNV 420RIDKVHLFVD QQWLAVRSKS NTNCGGGNHG YNNEFRSMEA IFLAHGPSFK EKTEVEPFEN 480IEVYNLMCDL LRIQPAPNNG THGSLNHLLK VPFYEPSHAE EVSKFSVCGF ANPLPTESLD 540CFCPHLQNST QLEQVNQMLN LTQEEITATV KVNLPFGRPR VLQKNVDHCL LYHREYVSGF 600GKAMRMPMWS SYTVPQLGDT SPLPPTVPDC LRADVRVPPS ESQKCSFYLA DKNITHGFLY 660PPASNRTSDS QYDALITSNL VPMYEEFRKM WDYFHSVLLI KHATERNGVN VVSGPIFDYN 720YDGHFDAPDE ITKHLANTDV PIPTHYFVVL TSCKNKSHTP ENCPGWLDVL PFIIPHRPTN 780VESCPEGKPE ALWVEERFTA HIARVRDVEL LTGLDFYQDK VQPVSEILQL KTYLPTFEIT 840 I841

TABLE LVIIIAmino acid sequence alignment of 161P2F10Bv.1 v.1 (SEQ ID NO: 101) and161P2F10B v.7 (SEQ ID NO: 102) Score = 1789 bits (4634), Expect =0.0 Identities = 838/841 (99%), Positives = 841/841 (99%) 161P2F10Bv.1:35 MSLGLGLGLGLRKLEKQGSCRKKCFDASFRGLENCRCDVACKDRGDCCWDFEDTCVESTR 94MSLGLGLGLGLRKLEKQGSCRKKCFDASFRGLENCRCDVACKDRGDCCWDFEDTCVESTR161P2F10Bv.7: 1MSLGLGLGLGLRKLEKQGSCRKKCFDASFRGLENCRCDVACKDRGDCCWDFEDTCVESTR 60161P2F10Bv.1: 95IWMCNKFRCGETRLEASLCSCSDDCLQKKDCCADYKSVCQGETSWLEENCDTAQQSQCPE 154IWMCNKFRCGETRLEASLCSCSDDCLQ+KDCCADYKSVCQGETSWLEENCDTAQQSQCPE1G1P2F10Bv.7: 61IWMCNKFRCGETRLEASLCSCSDDCLQRKDCCADYKSVCQGETSWLEENCDTAQQSQCPE 120161P2F10Bv.1: 155GFDLPPVILFSMDGFRAEYLYTWDTLMPNINKLKTCGIHSKYMRAMYPTKTFPNHYTIVT 214GFDLPPVILFSMDGFRAEYLYTWDTLMPNINKLKTCGIHSKYMRAMYPTKTFPNHYTIVT161P2F10Bv.7: 121GFDLPPVILFSMDGFRAEYLYTWDTLMPNINKLKTCGIHSKYMRAMYPTKTFPNHYTIVT 180161P2F10Bv.1: 215GLYPESHGIIDNNMYDVNLNKNFSLSSKEQNNPAWWHGQPMWLTAMYQGLKAATYFWPGS 274GLYPESHGIIDNNMYDVNLNKNFSLSSKEQNNPAWWHGQPMWLTAMYQGLKAATYFWPGS161P2F10Bv.7: 181GLYPESHGIIDNNMYDVNLNKNFSLSSKEQNNPAWWHGQPMWLTAMYQGLKAATYFWPGS 240161P2F10Bv.1: 275EVAINGSFPSIYMPYNGSVPFEERISTLLKWLDLPKAERPRFYTMYFEEPDSSGHAGGPV 334EVAINGSEPSIYMPYNGSVPFEERISTLLKWLDLPKAERPRFYTM+FEEPDSSGHAGGPV161P2F10Bv.7: 241EVAINGSFPSIYMPYNGSVPFEERISTLLKWLDLPKAERPRFYTMFFEEPDSSGHAGGPV 300161P2F10Bv.1: 335SARVIKALQVVDHAFGMLMEGLKQRNLHNCVNIILLADHGMDQTYCNKMEYMTDYFPRIN 394SARVIKALQVVDHAFGMLMEGLKQRNLHNCVNIILLADHGMDQTYCNKMEYMTDYFPRIN161P2F10Bv.7: 301SARVIKALQVVDHAFGMLMEGLKQRNLHNCVNIILLADHGMDQTYCNKMEYMTDYFPRIN 360161P2F10Bv.1: 395FFYMYEGPAPRTRAHNIPHDFFSFNSEEIVRNLSCRKPDQHFKPYLTPDLPKRLHYAKNV 454FFYMYEGPAPR+RAHNIPHDFFSFNSEEIVRNLSCRKPDQHFKPYLTPDLPKRLHYAKNV161P2F10Bv.7: 361FFYMYEGPAPRVRAHNIPHDFFSFNSEEIVRNLSCRKPDQHFKPYLTPDLPKRLHYAKNV 420161P2F10Bv.1: 455RIDKVHLFVDQQWLAVRSKSNTNCGGGNHGYNNEFRSMEAIFLAHGPSFKEKTEVEPFEN 514RIDKVHLFVDQQWLAVRSKSNTNCGGGNHGYNNEFRSMEAIFLAHGPSFKEKTEVEPFEN161P2F10Bv.7: 421RIDKVHLFVDQQWLAVRSKSNTNCGGGNHGYNNEFRSMEAIFLAHGPSFKEKTEVEPFEN 480161P2F10Bv.1: 515IEVYNLMCDLLRIQPAPNNGTHGSLNHLLKVPFYEPSHAEEVSKFSVCGFANPLPTESLD 574IEVYNLMCDLLRIQPAPNNGTHGSLNHLLKVPFYEPSHAEEVSKFSVCGFANPLPTESLD161P2F10Bv.7: 481IEVYNLMCDLLRIQPAPNNGTHGSLNHLLKVPFYEPSHAEEVSKFSVCGFANPLPTESLD 540161P2F10Bv.1: 575CFCPHLQNSTQLEQVNQMLNLTQEEITATVKVNLPFGRPRVLQKNVDHCLLYHREYVSGF 634CFCPHLQNSTQLEQVNQMLNLTQEEITATVKVNLPFGRPRVLQKNVDHCLLYHREYVSGF161P2F10Bv.7: 541CFCPHLQNSTQLEQVNQMLNLTQEEITATVKVNLPFGRPRVLQKNVDHCLLYHREYVSGF 600161P2F10Bv.1: 635GKAMRMPMWSSYTVPQLGDTSPLPPTVPDCLRADVRVPPSESQKCSFYLADKNITHGFLY 694GKAMRMPMWSSYTVPQLGDTSPLPPTVPDCLRADVRVPPSESQKCSFYLADKNITHGFLY161P2F10Bv.7: 601GKAMRMPMWSSYTVPQLGDTSPLPPTVPDCLRADVRVPPSESQKCSFYLADKNITHGFLY 660161P2F10Bv.1: 695PPASNRTSDSQYDALITSNLVPMYEEFRKMWDYFHSVLLIKHATERNGVNVVSGPIFDYN 754PPASNRTSDSQYDALITSNLVPMYEEFRKMWDYFHSVLLIKHATERNGVNVVSGPIFDYN161P2F10Bv.7: 661PPASNRTSDSQYDALITSNLVPMYEEFRKMWDYFHSVLLIKHATERNGVNVVSGPIFDYN 720161P2F10Bv.1: 755YDGHFDAPDEITKHLANTDVPIPTHYFVVLTSCKNKSHTPENCPGWLDVLPFIIPHRPTN 814YDGHFDAPDEITKHLANTDVPIPTHYFVVLTSCKNKSHTPENCPGWLDVLPFIIPHRPTN161P2F10Bv.7: 721YDGHFDAPDEITKHLANTDVPIPTHYFVVLTSCKNKSHTPENCPGWLDVLPFIIPHRPTN 780161P2F10Bv.1: 815VESCPEGKPEALWVEERFTAHIARVRDVELLTGLDFYQDKVQPVSEILQLKTYLPTFEIT 874VESCPEGKPEALWVEERFTAHIARVRDVELLTGLDFYQDKVQPVSEILQLKTYLPTFEIT161P2F10Bv.7: 781VESCPEGKPEALWVEERFTAHIARVRDVELLTGLDFYQDKVQPVSEILQLKTYLPTFEIT 840161P2F10Bv.1: 875 I 875 I 161P2F10Bv.7: 841 I 841

TABLE LIX 161P2F10B Expression in Kidney Cancer Transi- Onco- Clear cellPapillary Chromophobe tional cytoma RNA analysis: 33/34 (97%) 16/19(84%) 2/3 (67%) 3/7 (42%) 0/3 (0%) Protein analysis: 12/12 (100%)  5/5(100%) 1/3 (33%) 0/3 (0%) 0/2 (0%)

TABLE LX 161P2F10B protein expression in normal tissues TISSUE FREQUENCYKidney 5/5 Prostate 4/8 Bladder 1/4* Colon 2/5* Lung 1/4* Brain 0/1Breast 0/2 Heart 0/1 Liver 0/3 Ovary 0/1 Pancreas 0/2 Placenta 0/1 Skin0/1 Spleen 0/1 Testis 0/4 Thymus 0/1 Uterus 0/1

1. A method to detect cells expressing a 161P2F10B protein in aperipheral blood sample, comprising: contacting cells in the peripheralblood sample with an antibody or antigen-binding fragment thereof thatspecifically binds to said protein, wherein the protein consists of theamino acid sequence of SEQ ID NO:3, 17, 18, 19, or 20; and detectingbinding of said antibody or antigen-binding fragment thereof to saidcells, whereby binding of said antibody or antigen-binding fragmentthereof to said cells indicates that the cells express the protein,wherein said cells are kidney tumor cells.
 2. The method of claim 1,wherein the isolated antibody or antigen-binding fragment thereofimmunospecifically binds to an epitope from the amino acid sequence ofSEQ ID NO: 3, 17, 18, 19, or
 20. 3. The method of claim 1, wherein theantibody is a polyclonal antibody.
 4. The method of claim 1, wherein theantibody is a monoclonal antibody.
 5. The method of claim 4, wherein themonoclonal antibody is a recombinant protein.
 6. The method of claim 5,wherein the antibody is a single chain monoclonal antibody.
 7. Themethod of claim 1, wherein the fragment is an Fab, F(ab′)₂, Fv or Sfvfragment.